Highly-purified soluble thrombomodulin and method for producing same

ABSTRACT

Highly-purified soluble thrombomodulin which has a content of host cell-originated proteins being in a ratio of less than 10 ng of the proteins per 10,000 U of the soluble thrombomodulin, wherein the soluble thrombomodulin is produced by a transformant cell obtained by transfecting a host cell with a DNA containing a nucleotide sequence encoding the soluble thrombomodulin.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a Divisional of copending application Ser. No.13/641,047, filed on Dec. 21, 2012, which was filed as PCT InternationalApplication No. PCT/JP2011/060348 on Apr. 28, 2011, which claims thebenefit under 35 U.S.C. §119(a) to Patent Application No. 2010-105421,filed in Japan on Apr. 30, 2010, all of which are hereby expresslyincorporated by reference into the present application.

TECHNICAL FIELD

The present invention relates to highly-purified soluble thrombomodulinand a method for producing the same.

BACKGROUND ART

Thrombomodulin is known as a substance having an action of specificallybinding to thrombin to inhibit the blood coagulation activity ofthrombin, and at the same time, significantly promote the ability ofthrombin to activate Protein C, and is also known to have strong bloodcoagulation-inhibiting action. It is also known that thrombomodulinextends the thrombin clotting time, and that it suppresses plateletaggregation by thrombin. Protein C is a vitamin K-dependent protein thatplays an important role in the blood coagulation fibrinolytic system,and is activated by the action of thrombin to become activated ProteinC. It is known that the activated Protein C inactivates activated bloodcoagulation factor V and activated blood coagulation factor VIII invivo, and that it is involved in generation of plasminogen activatorhaving thrombolytic action (Non-patent document 1). Therefore,thrombomodulin is considered to be useful as an anticoagulant agent or athrombolytic agent that promotes the activation of Protein C bythrombin, and there have also been reported animal experimentsdemonstrating that thrombomodulin is effective for therapeutic andprophylactic treatments of diseases associated with acceleration ofcoagulation (Non-patent document 2).

Thrombomodulin was first discovered and obtained as a glycoproteinexpressed on vascular endothelial cells of various animal speciesincluding human, and thereafter successfully cloned. More specifically,a human thrombomodulin precursor gene containing a signal peptide wascloned from a human lung cDNA library by genetic engineering techniques,and the entire gene sequence of thrombomodulin was analyzed, so that theamino acid sequence consisting of 575 residues containing a signalpeptide (usually 18 amino acid residues are exemplified) was elucidated(Patent document 1). It is known that mature thrombomodulin, from whichthe signal peptide has been cleaved, is constituted by 5 regions,namely, an N-terminal region (amino acids 1 to 226, these positions areindicated on the assumption that the signal peptide consists of 18 aminoacid residues, and the same shall apply to the other regions), a regionhaving six EGF-like structures (amino acids 227 to 462), an O-linkedglycosylation region (amino acids 463 to 498), a transmembrane region(amino acids 499 to 521), and an intracytoplasmic region (amino acids522 to 557), from the N-terminal side of the mature peptide, and that apart having the same activity as that of the full length thrombomodulin(i.e., minimum unit for the activity) mainly consists of the 4th, 5th,and 6th EGF-like structure portions from the N-terminal side among thesix EGF-like structures (Non-patent document 3).

Unless a surfactant is present, the full length thrombomodulin is hardlydissolved, and therefore addition of a surfactant is essential forproducing a thrombomodulin preparation. In contrast, there is alsosoluble thrombomodulin that can be fully dissolved even in the absenceof a surfactant. The soluble thrombomodulin may be prepared so as not tocontain at least a part of the transmembrane region or the entiretransmembrane region. For example, it has been confirmed that a solublethrombomodulin consisting of only 3 regions of the N-terminal region,the region having six EGF-like structures, and the O-linkedglycosylation region (i.e., soluble thrombomodulin having an amino acidsequence comprising amino acids at the positions 19 to 516 in SEQ ID NO:1), can be obtained by applying recombination techniques, and that suchrecombinant soluble thrombomodulin has the same activity as that of thenatural thrombomodulin (Patent document 1). In addition, there are alsosome other reports regarding soluble thrombomodulin (Patent documents 2to 9). Further, human urine-derived soluble thrombomodulin and the likeare also exemplified as natural thrombomodulin (Patent documents 10 and11).

As recognized in many cases, as a result of spontaneous mutations ormutations occurring at the time of obtaining thrombomodulin, polymorphicmutations have been found even in human genes, and at present, suchthrombomodulin genes that the amino acid at the position 473 of thehuman thrombomodulin precursor, that has the aforementioned amino acidsequence consisting of 575 amino acid residues, is Val or Ala have beenidentified. This difference corresponds to the difference of thenucleotide at the position 1418 to T or C in the nucleotide sequencesencoding the amino acid (Non-patent document 4). However, these twothrombomodulins are completely identical in terms of their activitiesand physical properties. Thus, it can be considered that they aresubstantially identical.

It has been reported that thrombomodulin is effective for a therapeutictreatment of DIC (Non-patent documents 5 and 6). As for use ofthrombomodulin, in addition to the aforementioned uses, thrombomodulinis expected to be used in therapeutic and prophylactic treatments ofvarious diseases such as acute coronary syndrome (ACS), thrombosis,peripheral vessel obstruction, obstructive arteriosclerosis, vasculitis,functional disorder occurring after heart surgery, complication causedby organ transplantation, angina pectoris, transient ischemic attack,toxemia of pregnancy, diabetes, liver VOD (liver veno-occlusive disease,e.g., fulminant hepatitis, veno occlusive disease of liver occurringafter bone marrow transplantation), and deep venous thrombosis (DVT),and further, adult respiratory distress syndrome (ARDS).

As a premise of application of thrombomodulin in pharmaceuticalproducts, it is needless to explain that the soluble thrombomodulin isrequired to be manufactured in a large scale and at a cost as low aspossible. However, there is also pointed out a possibility thatheterogenous proteins originated in the production process, for example,proteins originated in host cells, bovine serum proteins originated inmedium, and mouse IgG and the like originated in antibody column serveas immunogens to case problems concerning safety (Non-patent document7).

As methods for producing soluble thrombomodulin in an industrial scalefor application as a pharmaceutical product, there are known, forexample, a method of using affinity column chromatography in a mainpurification step to which an antibody that reacts with thrombomodulinis bound, a method for producing highly purified soluble thrombomodulinsubstantially free from serum-originated substances andantibody-originated substances, which is characterized in that thesoluble thrombomodulin is obtained as a flow-through fraction in a stepof bringing the soluble thrombomodulin obtained by affinity columnchromatography into contact with a cation exchanger under conditions ofa specific conductivity of 25 to 34 ms/cm and pH 3 to 4 (Patent document12), and a method for purifying thrombomodulin, wherein affinity columnchromatography as the main purification step is followed by strong anionexchange chromatography (Patent document 13).

PRIOR ART REFERENCES Patent Documents

-   Patent document 1: Japanese Patent Unexamined Publication (Kokai)    No. 64-6219-   Patent document 2: Japanese Patent Unexamined Publication No.    2-255699-   Patent document 3: Japanese Patent Unexamined Publication No.    3-133380-   Patent document 4: Japanese Patent Unexamined Publication No.    3-259084-   Patent document 5: Japanese Patent Unexamined Publication No.    4-210700-   Patent document 6: Japanese Patent Unexamined Publication No.    5-213998-   Patent document 7: WO92/00325-   Patent document 8: WO92/03149-   Patent document 9: WO93/15755-   Patent document 10: Japanese Patent Unexamined Publication No.    3-86900-   Patent document 11: Japanese Patent Unexamined Publication No.    3-218399-   Patent document 12: Japanese Patent Unexamined Publication No.    11-341990-   Patent document 13: WO2008/117735

Non-Patent Documents

-   Non-patent document 1: Koji Suzuki, Igaku no Ayumi (Progress of    Medicine), Vol. 125, 901 (1983)-   Non-patent document 2: K. Gomi et al., Blood, 75, 1396-1399 (1990)-   Non-patent document 3: M. Zushi et al., J. Biol. Chem., 264,    10351-10353 (1989)-   Non-patent document 4: D. Z. Wen et al., Biochemistry, 26, 4350-4357    (1987)-   Non-patent document 5: S. M. Bates et al., Br. J. Pharmacol., 144,    1017-1028 (2005)-   Non-patent document 6: H. Saito et al., J. Thromb Haemost, 5 (1), 31    (2007)-   Non-patent document 7: Akio Hayakawa, Development and Security of    Quality and Safety of Biomedical Products, 273-274 (2007)

SUMMARY OF THE INVENTION Object to be Achieved by the Invention

An object of the present invention is to provide highly-purified solublethrombomodulin in which a concentration of proteins originated in hostcells is a ratio of less than 10 ng of the proteins per 10,000 U ofsoluble thrombomodulin, and a method for producing the same.

Means for Achieving the Object

Patent document 13 describes purified soluble thrombomodulin. Inparticular, it discloses soluble thrombomodulin in which concentrationof proteins originated in the host (henceforth also abbreviated as “HCP”in the specification) is indicated as “N.D.” (this indication seems tomean “not detected”, although it is not specifically indicated) inExample 14. Those skilled in the art who read this description wouldhave normally considered that highly-purified soluble thrombomodulincontaining reduced contamination of HCP was satisfactorily achieved, andwould not have considered to further reduce HCP in solublethrombomodulin, in other words, the artisans would not have beenmotivated to further reduce HCP in soluble thrombomodulin.

However, when the purified soluble thrombomodulin is used as apharmaceutical product, the HCP, if contaminated in the product, mightpossibly cause unexpected condition such as anaphylactic shock, andmight lead to a lethal risk, which the inventors of the presentinvention strongly recognized as a serious problem. From this reason,even under the circumstance that those skilled in the art would havenormally considered that a contamination of HCP was sufficiently reducedas Patent document 13 mentioned above describes “N.D.” in Example 14,the inventors of the present invention conducted measurement of a HCPconcentration in the purified soluble thrombomodulin described inExample 14 of Patent document 13 mentioned above. As a result, althoughthe concentration was almost near the detection limit, the contaminatedHCP concentration was found to be a ratio of less than 70 to 80 ng per10,000 U of the soluble thrombomodulin (henceforth “U” means a unit ofthe action for promoting activation of Protein C (henceforth alsoabbreviated as APC activity) as later described in Reference Example 1,unless otherwise specifically indicated), and thus the inventors of thepresent invention first recognized that improvement of the reduction ofHCP might still be possibly achievable. The inventors of the presentinvention themselves consider that the HCP concentration can beaccurately measured by employing an additional step of concentration inthe HCP measurement step, which additional step is not disclosed inPatent document 13.

The inventors of the present invention who first discovered theaforementioned fact found a novel object to obtain purifiedthrombomodulin consisting of the soluble thrombomodulin having a furtherreduced HCP content to minimize the risk of anaphylactic shock, with theaim of safer use of soluble thrombomodulin as a pharmaceutical product.

The inventors of the present invention studied an application of anadditional column chromatography step for industrial scale production ofhighly-purified soluble thrombomodulin in which contamination of HCP wasfurther reduced. Specifically, they tried to reduce HCP by combining aplurality of column chromatography steps with affinity columnchromatography considered to have the highest HCP-removing effect.However, such additional column chromatography steps not only increasedthe time and labor for the production, but also caused a problem ofreduction of the yield of soluble thrombomodulin. Moreover, even if aplurality of column chromatography steps were combined with affinitycolumn chromatography, highly-purified soluble thrombomodulin was notobtained in which the HCP concentration was further reduced comparedwith the conventional level. Furthermore, there also arose a problemthat, in the column chromatography, only a small change of pH, ionicstrength or the like resulted in a change of separation of HCP andsoluble thrombomodulin, and thus an expected result was not successfullyreproduced. Accordingly, it was difficult to obtain highly-purifiedsoluble thrombomodulin.

Therefore, the inventors of the present invention conducted variousresearches to find a method for obtaining highly-purified solublethrombomodulin containing further reduced contamination of HCP byefficiently eliminating HCP at an industrially acceptable level withoutreducing the yield of soluble thrombomodulin with a simpler operationcompared with column chromatography. As a result, they found that theaforementioned object of obtaining highly-purified solublethrombomodulin having an HCP concentration corresponding to a ratio ofless than 10 ng of HCP per 10,000 U of soluble thrombomodulin wassuccessfully achieved by using nylon and/or polyethersulfone, inparticular, by using nylon, and thus accomplished the present invention.

The present invention is thus embodied as follows.

[1] Highly-purified soluble thrombomodulin which has a content of hostcell-originated proteins being in a ratio of less than 10 ng of theproteins per 10,000 U of the soluble thrombomodulin, wherein the solublethrombomodulin is produced by a transformant cell obtained bytransfecting a host cell with a DNA containing a nucleotide sequenceencoding the soluble thrombomodulin.[1-2] The highly-purified soluble thrombomodulin according to [1]mentioned above, wherein purity of the highly-purified solublethrombomodulin is 99% or higher based on the total proteins.[1-3] The highly-purified soluble thrombomodulin according to [1] or[1-2] mentioned above, wherein the highly-purified solublethrombomodulin is in the form of an aqueous solution.[1-4] The highly-purified soluble thrombomodulin according to [1-3]mentioned above, wherein a concentration of soluble thrombomodulin inthe aqueous solution of the highly-purified soluble thrombomodulin is 8mg/mL or higher.[2] The highly-purified soluble thrombomodulin according to any one of[1] to [1-4] mentioned above, wherein the soluble thrombomodulin isthrombomodulin produced by serum-free culture of the transformant cell.

When the referred item numbers are indicated with such a range as “[1]to [1-4]” mentioned above, and the range includes an item indicated witha number having a subnumber such as [1-2], it is meant that the itemindicated with the number having a subnumber such as [1-2] is alsocited. The same shall apply to the following definitions.

[2-2] The highly-purified soluble thrombomodulin according to any one of[1] to [2] mentioned above, wherein the concentration of hostcell-originated proteins of less than 10 ng per 10,000 U of solublethrombomodulin is confirmed by measuring content of the hostcell-originated proteins by a method comprising at least the followingsteps:(a) the step of preparing host cell-originated proteins from culturesupernatant obtained by carrying out serum-free culture of atransformant cell obtained by transfecting the host cell with a DNAcontaining a nucleotide sequence encoding the soluble thrombomodulin, orthe host cell,(b) the step of purifying an anti-host cell-originated protein antibodyfrom antiserum obtained by sensitizing a rabbit with the hostcell-originated proteins obtained in (a) mentioned above, the step ofconstructing a measurement system comprising:(c1) the step of adsorbing the anti-host cell-originated proteinantibody obtained in (b) mentioned above to a solid phase,(c2-1) the step of bringing a soluble thrombomodulin-containing testsolution suspected to be contaminated with the host cell-originatedproteins into contact with the solid phase to which the anti-hostcell-originated protein antibody is adsorbed, and the step of bringing asolution containing the host cell-originated proteins of a knownconcentration into contact with the solid phase to which the anti-hostcell-originated protein antibody is adsorbed,(c3) the step of adding a biotinylated anti-host cell-originated proteinantibody to the solid phase,(c4) the step of adding a solution of avidinylated peroxidase to thesolid phase,(c5) the step of adding an enzyme substrate solution to allow colordevelopment, and(c6) the step of terminating the color development and measuringabsorbance,(d) the step of measuring concentration of the host cell-originatedproteins in the soluble thrombomodulin-containing test solutionsuspected to be contaminated with the host cell-originated proteins inthe aforementioned measurement system, and determining whether theconcentration of the host cell-originated proteins in the solublethrombomodulin-containing test solution is within a range that enablesquantification of the proteins in the aforementioned measurement system,which range is confirmed beforehand by performing measurement using asolution of the host cell-originated proteins of a known concentrationin the aforementioned measurement system,(e-1) the step of determining the concentration of the hostcell-originated proteins in the soluble thrombomodulin-containing testsolution suspected to be contaminated with the host cell-originatedproteins as the concentration of the host cell-originated proteins inthe solution, when the concentration is determined to be within therange that enables the quantification in (d) mentioned above,(e-2-1) the step of concentrating or diluting the solublethrombomodulin-containing test solution suspected to be contaminatedwith the host cell-originated proteins, if desired, to make theconcentration of the host cell-originated proteins to be a measurableconcentration within the range that enables the quantification in theaforementioned measurement system, when the concentration of the hostcell-originated proteins is determined to be not within the range thatenables the quantification in (d) mentioned above, and recording theconcentration ratio or dilution ratio,(e-2-2) the step of measuring the host cell-originated proteinconcentration in the soluble thrombomodulin-containing test solutionconcentrated or diluted in (e-2-1) mentioned above in a measurementsystem corresponding to the measurement system represented by the stepsof (c1) to (c6) mentioned above in which (c2-1) is replaced with (c2-2)mentioned below, and obtaining the host cell-originated proteinconcentration with taking the concentration ratio or dilution ratio intoconsideration,(c2-2) the step of bringing the soluble thrombomodulin-containing testsolution concentrated or diluted, if necessary, into contact with thesolid phase to which the anti-host cell-originated protein antibody isadsorbed, and the step of bringing a solution containing the hostcell-originated proteins of a known concentration into contact with thesolid phase to which the anti-host cell-originated protein antibody isadsorbed, and(f) the step of calculating ratio of the host cell-originated proteinconcentration obtained in (e-1) or (e-2-2) based on APC activity ofthrombomodulin per unit volume of the soluble thrombomodulin-containingtest solution measured separately.[2-3] The highly-purified soluble thrombomodulin according to [2-2]mentioned above, wherein the host cell mentioned in [2-2], (a) mentionedabove is a cell of Chinese hamster ovary cell line DXB11.[3] The highly-purified soluble thrombomodulin according to any one of[1] to [2-3] mentioned above, wherein the host cell is a Chinese hamsterovary cell.[4] The highly-purified soluble thrombomodulin according to any one of[1] to [3] mentioned above, wherein the soluble thrombomodulin has thefollowing properties (1) to (5):(1) an action of selectively binding to thrombin,(2) an action of promoting activation of Protein C by thrombin,(3) an action of extending thrombin clotting time,(4) an action of suppressing platelet aggregation caused by thrombin,and(5) anti-inflammatory action.[4-2] The highly-purified soluble thrombomodulin according to any one of[1] to [3] mentioned above, wherein the soluble thrombomodulin has thefollowing properties (1) to (4):(1) an action of selectively binding to thrombin,(2) an action of promoting activation of Protein C by thrombin,(3) an action of extending thrombin clotting time, and(4) an action of suppressing platelet aggregation caused by thrombin.[5] The highly-purified soluble thrombomodulin according to any one of[1] to [4-2] mentioned above, wherein molecular weight of the solublethrombomodulin is 50,000 to 80,000.[6] The highly-purified soluble thrombomodulin according to any one of[1] to [5] mentioned above, wherein the highly-purified solublethrombomodulin is produced by a method comprising the following steps:(a) the step of obtaining a transformant cell by transfecting a hostcell with a DNA encoding a soluble thrombomodulin;(b) the step of obtaining a solution containing the solublethrombomodulin by culturing the transformant cell, and(c) the step of bringing the solution containing the solublethrombomodulin into contact with nylon and/or polyethersulfone to obtainhighly-purified soluble thrombomodulin having a content of hostcell-originated proteins being in a ratio of less than 10 ng of theproteins per 10,000 U of soluble thrombomodulin.[7] The highly-purified soluble thrombomodulin according to any one of[1] to [6] mentioned above, wherein the soluble thrombomodulin is apeptide containing:(i) the amino acid sequence of the positions 367 to 480 in the aminoacid sequence of SEQ ID NO: 9 or 11, and the amino acid sequence of(ii-1) or (ii-2) mentioned below, and the peptide is solublethrombomodulin having the following properties (1) to (5):(ii-1) the amino acid sequence of the positions 19 to 244 in the aminoacid sequence of SEQ ID NO: 9 or 11, or(ii-2) the amino acid sequence of (ii-1) mentioned above, furtherincluding substitution, deletion or addition of one or more amino acidresidues,(1) an action of selectively binding to thrombin,(2) an action of promoting activation of Protein C by thrombin,(3) an action of extending thrombin clotting time,(4) an action of suppressing platelet aggregation caused by thrombin,and(5) anti-inflammatory action.[7-2] The highly-purified soluble thrombomodulin according to any one of[1] to [6] mentioned above, wherein the soluble thrombomodulin is apeptide containing:(i) the amino acid sequence of the positions 367 to 480 in the aminoacid sequence of SEQ ID NO: 9 or 11, and the amino acid sequence of(ii-1) or (ii-2) mentioned below, and the peptide is solublethrombomodulin having the following properties (1) to (4):(ii-1) the amino acid sequence of the positions 19 to 244 in the aminoacid sequence of SEQ ID NO: 9 or 11, or(ii-2) the amino acid sequence of (ii-1) mentioned above, furtherincluding substitution, deletion or addition of one or more amino acidresidues,(1) an action of selectively binding to thrombin,(2) an action of promoting activation of Protein C by thrombin,(3) an action of extending thrombin clotting time, and(4) an action of suppressing platelet aggregation caused by thrombin.[7-3] The highly-purified soluble thrombomodulin according to any one of[1] to [6] mentioned above, wherein the soluble thrombomodulin is apeptide containing the amino acid sequence of (i-1) or (i-2) mentionedbelow, and containing the amino acid sequence of (ii-1) or (ii-2)mentioned below, and the peptide is soluble thrombomodulin having theproperties (1) to (5) mentioned below:(i-1) the amino acid sequence of the positions 367 to 480 in the aminoacid sequence of SEQ ID NO: 9 or 11, or(i-2) the amino acid sequence of (i-1) mentioned above, furtherincluding substitution, deletion or addition of one or more amino acidresidues,(ii-1) the amino acid sequence of the positions 19 to 244 in the aminoacid sequence of SEQ ID NO: 9 or 11, or(ii-2) the amino acid sequence of (ii-1) mentioned above, furtherincluding substitution, deletion or addition of one or more amino acidresidues,(1) an action of selectively binding to thrombin,(2) an action of promoting activation of Protein C by thrombin,(3) an action of extending thrombin clotting time,(4) an action of suppressing platelet aggregation caused by thrombin,and(5) anti-inflammatory action.[8] The highly-purified soluble thrombomodulin according to any one of[1] to [6] mentioned above, wherein the soluble thrombomodulin is apeptide containing:(i-1) the amino acid sequence of the positions 19 to 516 in the aminoacid sequence of SEQ ID NO: 9 or 11, or(i-2) the amino acid sequence of (i-1) mentioned above, furtherincluding substitution, deletion or addition of one or more amino acidresidues, and the peptide is soluble thrombomodulin having theproperties (1) to (5) mentioned below:(1) an action of selectively binding to thrombin,(2) an action of promoting activation of Protein C by thrombin,(3) an action of extending thrombin clotting time,(4) an action of suppressing platelet aggregation caused by thrombin,and(5) anti-inflammatory action.[9] The highly-purified soluble thrombomodulin according to any one of[1] to [6] mentioned above, wherein the DNA containing a nucleotidesequence encoding soluble thrombomodulin is a DNA encoding the aminoacid sequence of SEQ ID NO: 9 or 11.[10] A pharmaceutical composition containing the highly-purified solublethrombomodulin according to any one of [1] to [9] mentioned above and apharmaceutically acceptable carrier.[11] A method for preparing highly-purified soluble thrombomodulinhaving a content of host cell-originated proteins being in a ratio ofless than 10 ng of the proteins per 10,000 U of soluble thrombomodulin,which comprises the step of bringing a solution containing solublethrombomodulin produced by a transformant cell obtained by transfectinga host cell with a DNA containing a nucleotide sequence encoding solublethrombomodulin into contact with nylon and/or polyethersulfone.[12] The preparation method according to [11] mentioned above, whereinthe soluble thrombomodulin is prepared by serum-free culture of thetransformant cell.[13] The method for preparing highly-purified soluble thrombomodulinaccording to [11] or [12] mentioned above, wherein the solublethrombomodulin has the following properties (1) to (5);(1) an action of selectively binding to thrombin,(2) an action of promoting activation of Protein C by thrombin,(3) an action of extending thrombin clotting time,(4) an action of suppressing platelet aggregation caused by thrombin,and(5) anti-inflammatory action.[14] The preparation method according to any one of [11] to [13]mentioned above, wherein the host cell is a Chinese hamster ovary cell.[15] The preparation method according to any one of [11] to [14]mentioned above, wherein molecular weight of the soluble thrombomodulinis 50,000 to 80,000.[16] The preparation method according to any one of [11] to [15]mentioned above, wherein the soluble thrombomodulin is a peptidecontaining:(i) the amino acid sequence of the positions 367 to 480 in the aminoacid sequence of SEQ ID NO: 9 or 11, and the amino acid sequence of(ii-1) or (ii-2) mentioned below, and the peptide is solublethrombomodulin having the following properties (1) to (5):(ii-1) the amino acid sequence of the positions 19 to 244 in the aminoacid sequence of SEQ ID NO: 9 or 11, or(ii-2) the amino acid sequence of (ii-1) mentioned above, furtherincluding substitution, deletion or addition of one or more amino acidresidues,(1) an action of selectively binding to thrombin,(2) an action of promoting activation of Protein C by thrombin,(3) an action of extending thrombin clotting time,(4) an action of suppressing platelet aggregation caused by thrombin,and(5) anti-inflammatory action.[17] The preparation method according to any one of [11] to [15]mentioned above, wherein the soluble thrombomodulin is a peptidecontaining:(i-1) the amino acid sequence of the positions 19 to 516 in the aminoacid sequence of SEQ ID NO: 9 or 11, or(i-2) the amino acid sequence of (i-1) mentioned above, furtherincluding substitution, deletion or addition of one or more amino acidresidues, andthe peptide is soluble thrombomodulin having the properties (1) to (5)mentioned below:(1) an action of selectively binding to thrombin,(2) an action of promoting activation of Protein C by thrombin,(3) an action of extending thrombin clotting time,(4) an action of suppressing platelet aggregation caused by thrombin,and(5) anti-inflammatory action.[18] The preparation method according to any one of [11] to [15]mentioned above, wherein the DNA containing a nucleotide sequenceencoding soluble thrombomodulin is a DNA encoding the amino acidsequence of SEQ ID NO: 9 or 11.[19] The preparation method according to any one of [11] to [18]mentioned above, wherein the nylon and/or polyethersulfone is in theform of a filtration membrane.[20] The preparation method according to [19] mentioned above, whereinthe filtration membrane has a membrane area of 0.01 to 0.5 m² for 1 mgof the host cell-originated proteins.[21] The preparation method according to any one of [11] to [20]mentioned above, wherein the nylon and/or polyethersulfone is nylon.[22] The preparation method according to any one of [11] to [20]mentioned above, which has the characteristics described in any one of[1-2] to [1-4], [2-2], [2-3], [7-2] and [7-3].[23] The preparation method according to any one of [11] to [22]mentioned above, which is a method for preparing highly-purified solublethrombomodulin having a content of host cell-originated proteins beingin a ratio of less than 10 ng of the proteins per 10,000 U of solublethrombomodulin, comprising:(a) the step of obtaining a transformant cell by transfecting a hostcell with a DNA encoding the amino acid sequence of SEQ ID NO: 9 or 11,(b) the step of obtaining a solution containing soluble thrombomodulinby culturing the transformant cell,(c) the step of purifying the solution containing soluble thrombomodulinto obtain a thrombomodulin purity of 99% or higher based on the totalproteins, and(d) the step of bringing the solution containing soluble thrombomodulininto contact with nylon to isolate highly-purified solublethrombomodulin having a content of host cell-originated proteins beingin a ratio of less than 10 ng of the proteins per 10,000 U of solublethrombomodulin, andwherein the host cell is a Chinese hamster ovary cell.[24] Highly-purified soluble thrombomodulin producible by the methodaccording to [23] mentioned above.[25] A method for removing host cell-originated proteins in solublethrombomodulin, which comprises the step of bringing a solutioncontaining soluble thrombomodulin produced by a transformant cellobtained by transfecting a host cell with a DNA containing a nucleotidesequence encoding soluble thrombomodulin into contact with nylon and/orpolyethersulfone.[26] A method for removing host cell-originated proteins in solublethrombomodulin, which comprises:(a) the step of obtaining a transformant cell by transfecting a hostcell with a DNA encoding the amino acid sequence of SEQ ID NO: 9 or 11,(b) the step of obtaining a solution containing soluble thrombomodulinby culturing the transformant cell,(c) the step of purifying the solution containing soluble thrombomodulinto a thrombomodulin purity of 99% or higher based on the total proteins,and(d) the step of bringing the solution containing soluble thrombomodulininto contact with nylon, and wherein the host cell is a Chinese hamsterovary cell.[27] The method for removing host cell-originated proteins in solublethrombomodulin according to [25] mentioned above, which has thecharacteristics mentioned in any one of [1] to [9] mentioned above.

Effect of the Invention

By using the preparation method of the present invention,highly-purified soluble thrombomodulin of reduced contamination of hostcell-originated proteins having a content of host cell-originatedproteins being in a ratio of less than 10 ng of the proteins per 10,000U of soluble thrombomodulin can be obtained. The risk of anaphylacticshock at the time of using soluble thrombomodulin as a pharmaceuticalproduct can be thereby further reduced.

MODES FOR CARRYING OUT THE INVENTION

Hereafter, several preferred embodiments of the present invention(preferred modes for carrying out the invention, henceforth alsoreferred to as “embodiments” in this specification) will be specificallyexplained. However, the scope of the present invention is not limited tothe specific embodiments explained below.

The highly-purified soluble thrombomodulin of this embodiment can beused as a material for a medicament. The highly-purified solublethrombomodulin of this embodiment can also be combined with anotherpharmaceutically acceptable carrier and used as a pharmaceuticalproduct.

Further, the highly-purified soluble thrombomodulin of this embodimentcan be used as a highly-purified soluble thrombomodulin-containingpharmaceutical composition not substantially containing any substancesother than the soluble thrombomodulin. The highly-purified solublethrombomodulin of this embodiment can also be combined with anotherpharmaceutically acceptable carrier and used as a pharmaceuticalcomposition.

Hereafter, the term of highly-purified soluble thrombomodulin mayinclude highly-purified soluble thrombomodulin as a material for amedicament. The term of highly-purified soluble thrombomodulin may alsoinclude a highly-purified soluble thrombomodulin-containingpharmaceutical composition not substantially containing any substancesother than the soluble thrombomodulin.

The thrombomodulin of this embodiment preferably is known to have anaction of (1) selectively binding to thrombin (2) to promote activationof Protein C by thrombin. In addition, it is preferred that thethrombomodulin is confirmed to generally have (3) an action of extendingthrombin clotting time, (4) an action of suppressing plateletaggregation caused by thrombin, and/or (5) anti-inflammatory action.Such actions possessed by thrombomodulin may be referred to asthrombomodulin activities.

As the thrombomodulin activities, thrombomodulin preferably has theactions of (1) and (2) mentioned above, and more preferably has theactions of (1) to (4) mentioned above. As the thrombomodulin activities,thrombomodulin more preferably has all of the actions of (1) to (5)mentioned above.

The action of thrombomodulin to bind with thrombin can be confirmed bythe test methods described in various known publications such asThrombosis and Haemostasis, 70(3):418-422 (1993). As for the action ofpromoting activation of Protein C by thrombin, degree of the activity ofpromoting the activation of Protein C by thrombin or presence or absenceof the action can be easily confirmed by the test methods clearlydescribed in various known publications including, for example, JapanesePatent Unexamined Publication No. 64-6219. Further, the action ofextending thrombin clotting time, and/or the action of suppressingplatelet aggregation caused by thrombin can be similarly and easilyconfirmed. Furthermore, the anti-inflammatory action can also beconfirmed by the test methods described in various known publicationsincluding, for example, Blood, 112:3361-3670 (2008) and The Journal ofClinical Investigation, 115, 5:1267-1274 (2005).

An example of the soluble thrombomodulin includes a solublethrombomodulin that is soluble in water in the absence of a surfactant.For example, the solubility of soluble thrombomodulin is preferably 1mg/ml or higher, or 10 mg/ml or higher in water, for example, indistilled water used for injection (in general, around a neutral rangein the absence of a surfactant such as Triton X-100 or Polidocanol). Thesolubility is more preferably 15 mg/ml or higher, or 17 mg/ml or higher;still more preferably 20 mg/ml or higher, 25 mg/ml or higher, or 30mg/ml or higher; and mosty preferably 60 mg/ml or higher. In some cases,the solubility may be 80 mg/ml or higher, or 100 mg/ml or higher. Fordetermining whether or not soluble thrombomodulin can be dissolved, thesolution can be observed with the naked eye, for example, directly belowwhite light source, at a position of brightness of approximately 1,000lux after the soluble thrombomodulin is dissolved, and it can beunderstood that transparency of the resulting solution and nocontamination of apparently observable insoluble substances may be clearcriteria of dissolution. In addition, it is also possible to filtratethe solution to confirm the presence or absence of a residue.

The molecular weight of the soluble thrombomodulin is not limited so farthat it has the thrombomodulin activities and is soluble as describedabove. The molecular weight is preferably 100,000 or smaller, morepreferably 90,000 or smaller, still more preferably 80,000 or smaller,most preferably 70,000 or smaller, and the molecular weight ispreferably 50,000 or larger, most preferably 60,000 or larger. Themolecular weight of the soluble thrombomodulin can be easily measured byordinary methods for measuring molecular weight of protein. Measurementby mass spectrometry is preferred, and MALDI-TOF-MS method is morepreferred.

For obtaining soluble thrombomodulin having a molecular weight within adesired range, a soluble thrombomodulin, which is obtained by culturinga transformant cell prepared by transfecting a host cell with a DNAencoding soluble thrombomodulin using a vector, can be subjected tofractionation using column chromatography or the like as describedlater.

As the soluble thrombomodulin, those of the human-type thrombomodulinare preferred which include the amino acid sequence of the positions 19to 132 in SEQ ID NO: 1 together with the amino acid sequence of thepositions 19 to 244 in SEQ ID NO: 9 or 11 or said amino acid sequencefurther including substitution, deletion or addition of one or moreamino acid residues. The amino acid sequence of the positions 19 to 132in SEQ ID NO: 1 participates in the action of promoting activation ofProtein C by thrombin among the thrombomodulin activities. The aminoacid sequence of the positions 19 to 244 in SEQ ID NO: 9 or 11participates in the anti-inflammatory action among the thrombomodulinactivities. So far that the soluble thrombomodulin has thethrombomodulin activities as the whole soluble thrombomodulin, the aminoacid sequence of the positions 19 to 132 in SEQ ID NO: 1 may benaturally or artificially mutated, namely, the amino acid sequence ofthe positions 19 to 132 in SEQ ID NO: 1 may include substitution,deletion or addition of one or more amino acid residues. Acceptabledegree of the mutation is not particularly limited so far that thesoluble thrombomodulin has the thrombomodulin activities, for example,including homology of 50% or higher based on an amino acid sequence,preferably homology of 70% or higher, more preferably homology of 80% orhigher, further preferably homology of 90% or higher, further morepreferably homology of 95% or higher, and most preferably homology of98% or higher. Such mutated amino acid sequence including substitution,deletion or addition of one or more amino acid residues is referred toas homologous mutation sequence. As described later, these mutated aminoacid sequences can be easily produced by using ordinary genemanipulation techniques. The soluble thrombomodulin is not particularlylimited so far that it has the aforementioned sequence and the action ofselectively binding to thrombin to promote activation of Protein C bythrombin at least as the whole soluble thrombomodulin. The solublethrombomodulin preferably also has the anti-inflammatory action.

In the amino acid sequence of SEQ ID NO: 3, Val as the amino acid at theposition 125 in the amino acid sequence of SEQ ID NO: 1 is replaced byAla. It is also preferred that the thrombomodulin of this embodimentcontains the amino acid sequence of the positions 19 to 132 in SEQ IDNO: 3.

As described above, the soluble thrombomodulin is not particularlylimited, so far that it has at least the sequence of the positions 19 to132 in SEQ ID NO: 1 or 3, or a homologous mutation sequence thereof, andthe amino acid sequence of the positions 19 to 244 in SEQ ID NO: 9 or11, or a homologous mutation sequence thereof, and has at least theaction of selectively binding to thrombin to promote activation ofProtein C by thrombin as the whole soluble thrombomodulin. Preferredexamples include a peptide comprising the sequence of the positions 19to 132 or the positions 17 to 132 in SEQ ID NO: 1 or 3, or a homologousmutation sequence thereof, and the amino acid sequence of the positions19 to 244 in SEQ ID NO: 9 or 11 or a homologous mutation sequencethereof, and having the thrombomodulin activities at least as the wholesoluble thrombomodulin, and a peptide comprising the sequence of thepositions 19 to 132 in SEQ ID NO: 1 or 3 and the amino acid sequence ofthe positions 19 to 244 in SEQ ID NO: 9 or 11 or a homologous mutationsequence thereof is more preferred. There is also another embodiment inwhich a peptide comprising the sequence of the positions 19 to 132 orthe positions 17 to 132 in SEQ ID NO: 1 or 3 or a homologous mutationsequence thereof, and the amino acid sequence of the positions 19 to 244in SEQ ID NO: 9 or 11 or a homologous mutation sequence, and having thethrombomodulin activities at least as the whole soluble thrombomodulin,which is sometimes more preferred.

It is preferred that the soluble thrombomodulin also has theanti-inflammatory action as the whole soluble thrombomodulin.

As another embodiment, the thrombomodulin preferably contains the aminoacid sequence of the positions 19 to 480 in SEQ ID NO: 5, and suchthrombomodulin is not particularly limited so far that it contains theamino acid sequence of the positions 19 to 480 in SEQ ID NO: 5. Theamino acid sequence of the positions 19 to 480 in SEQ ID NO: 5 may be ahomologous mutation sequence so far that it has the thrombomodulinactivities.

The amino acid sequence of SEQ ID NO: 7 corresponds to the amino acidsequence of SEQ ID NO: 5 in which Val as the amino acid at the position473 is replaced with Ala. The soluble thrombomodulin of this embodimentalso preferably contains the amino acid sequence of the positions 19 to480 in SEQ ID NO: 7.

As described above, the soluble thrombomodulin is not particularlylimited so far that it has at least the amino acid sequence of thepositions 19 to 480 in SEQ ID NO: 5 or 7, or a homologous mutationsequence thereof, and contains a peptide sequence having at least thethrombomodulin activities. Preferred examples include a peptidecomprising the sequence of the positions 19 to 480 or the positions 17to 480 in SEQ ID NO: 5 or 7, or a homologous mutation sequence thereof,and having at least the thrombomodulin activities, and a peptidecomprising the sequence of the positions 19 to 480 in SEQ ID NO: 5 or 7is more preferred. There is also another embodiment in which a peptidecomprising a homologous mutation sequence of the sequence of thepositions 19 to 480 or the positions 17 to 480 in SEQ ID NO: 5 or 7 andhaving at least the thrombomodulin activity, which is sometime morespreferred.

It is preferred that the soluble thrombomodulin also has theanti-inflammatory action.

As another particularly preferred embodiment, the soluble thrombomodulinpreferably contains the amino acid sequence of the positions 19 to 515in SEQ ID NO: 9, and such soluble thrombomodulin is not particularlylimited so far that it contains the amino acid sequence of the positions19 to 515 in SEQ ID NO: 9. The amino acid sequence of the positions 19to 515 o in SEQ ID NO: 9 may be a homologous mutation sequence thereofso far that it has the thrombomodulin activities.

The amino acid sequence of SEQ ID NO: 11 corresponds to the amino acidsequence of SEQ ID NO: 9 in which Val as the amino acid at the position473 is replaced with Ala. The soluble thrombomodulin of this embodimentalso preferably contains the amino acid sequence of the positions 19 to515 in SEQ ID NO: 11.

As described above, the soluble thrombomodulin is not particularlylimited so far that it contains a peptide having at least the amino acidsequence of the positions 19 to 515 in SEQ ID NO: 9 or 11, or ahomologous mutation sequence thereof, and having at least thethrombomodulin activities. More preferred examples include a peptidecomprising the amino acid sequence of the positions 19 to 516, 19 to515, 19 to 514, 17 to 516, 17 to 515, or 17 to 514 in SEQ ID NO: 9 or11, or a peptide comprising a homologous mutation sequence thereof andhaving at least the thrombomodulin activities, and a peptide comprisingthe amino acid sequence of the positions 19 to 516, 19 to 515, 19 to514, 17 to 516, 17 to 515, or 17 to 514 in SEQ ID NO: 9 is particularlypreferred. In addition, a mixture thereof is also a preferred example.There is another preferred embodiment in which a peptide comprising theamino acid sequence of the positions 19 to 516, 19 to 515, 19 to 514, 17to 516, 17 to 515, or 17 to 514 in SEQ ID NO: 11, which is particularlypreferred. A mixture thereof is also a preferred example. Further, apeptide comprising a homologous mutation sequence thereof and having atleast the thrombomodulin activities is also another preferred example.

It is preferred that the soluble thrombomodulin also has theanti-inflammatory action.

A peptide having a homologous mutation sequence is as described above.Such a peptide having a homologous mutation sequence also includes apeptide that may include substitution, deletion, or addition of one ormore, namely, one or multiple, more preferably several (for example, 1to 20, preferably 1 to 10, more preferably 1 to 5, most preferably 1 to3) amino acid residues in the amino acid sequence of the target peptide.Acceptable degree of mutation is not particularly limited so far thatthe peptide has the thrombomodulin activities. Examples include, forexample, homology of 50% or higher, preferably homology of 70% orhigher, more preferably homology of 80% or higher, further preferablyhomology of 90% or higher, further more preferably homology of 95% orhigher, and most preferably homology of 98% or higher based on an aminoacid sequence.

As the soluble thrombomodulin, preferred examples further include apeptide comprising the sequence of SEQ ID NO: 14 (462 amino acidresidues), a peptide comprising the sequence of SEQ ID NO: 8 (272 aminoacid residues), and a peptide comprising the sequence of SEQ ID NO: 6(236 amino acid residues) described in Japanese Patent UnexaminedPublication No. 64-6219.

The soluble thrombomodulin is not particularly limited so far that it isa peptide having at least the sequence of the positions 19 to 132 in SEQID NO: 1 or 3, or a homologous mutation sequence thereof, and the aminoacid sequence of the positions 19 to 244 in SEQ ID NO: 9 or 11, or ahomologous mutation sequence thereof, and has the thrombomodulinactivities at least as the whole soluble thrombomodulin. A peptidecomprising at least the amino acid sequence of the positions 19 to 480in SEQ ID NO: 5 or 7 is preferred, and a peptide comprising at least theamino acid sequence of the positions 19 to 515 in SEQ ID NO: 9 or 11 ismore preferred. More preferred examples of the peptide comprising atleast the amino acid sequence of the positions 19 to 515 in SEQ ID NO: 9or 11 include a peptide comprising the amino acid sequence of thepositions 19 to 516, 19 to 515, 19 to 514, 17 to 516, 17 to 515, or 17to 514 in SEQ ID NO: 9 or 11. More preferred examples also include amixture of peptides comprising the amino acid sequence of the positions19 to 516, 19 to 515, 19 to 514, 17 to 516, 17 to 515, or 17 to 514 foreach of SEQ ID NOS: 9 and 11.

In the case of the aforementioned mixture, the mixing ratio of a peptidethat starts from the position 17 and a peptide that starts from theposition 19 for each of SEQ ID NOS: 9 and 11 is, for example, 30:70 to50:50, preferably 35:65 to 45:55.

Further, the mixing ratio of a peptide that terminates at the position514, a peptide that terminates at the position 515, and a peptide thatterminates at the position 516 for each of SEQ ID NOS: 9 and 11 is, forexample, 0:0:100 to 0:90:10, or 0:70:30 to 10:90:0, or 10:0:90 to20:10:70, if desired.

The mixing ratio of the peptides can be determined by an ordinarymethod.

The sequence of the positions 19 to 132 in SEQ ID NO: 1 corresponds tothe sequence of the positions 367 to 480 in SEQ ID NO: 9, and thesequence of the positions 19 to 480 in SEQ ID NO: 5 corresponds to thesequence of the positions 19 to 480 in SEQ ID NO: 9.

Further, the sequence of the positions 19 to 132 in SEQ ID NO: 3corresponds to the sequence of the positions 367 to 480 in SEQ ID NO:11, and the sequence of the positions 19 to 480 in SEQ ID NO: 7corresponds to the sequence of the positions 19 to 480 in SEQ ID NO: 11.

Furthermore, all the sequences of the positions 1 to 18 in SEQ ID NOS:1, 3, 5, 7, 9 and 11 are identical sequences.

These soluble thrombomodulin can be obtained, for example, from atransformant cell prepared by transfecting a host cell with a DNAencoding any of those peptides (specifically, a nucleotide sequencehaving the nucleotide sequence of the positions 1 to 732 in SEQ ID NO:10 and the nucleotide sequence of the positions 55 to 396 in SEQ ID NO:2, a nucleotide sequence having the nucleotide sequence of the positions1 to 732 in SEQ ID NO: 10 and the nucleotide sequence of the positions55 to 396 in SEQ ID NO: 4, a nucleotide sequence of SEQ ID NO: 6, 8, 10or 12) using a vector, as described later.

It is sufficient that these peptides have any of the aforementionedamino acid sequences. A sugar chain may be or may not be added, and thepeptides are not particularly limited in this respect. In addition, ingene manipulation, type of such a sugar chain, position to which a sugarchain is added, and degree of addition may vary depending on the type ofthe host cell used, and they are not particularly limited. The bindingposition of such a sugar chain and the type thereof are described inJapanese Patent Unexamined Publication No. 11-341990, and in the case ofthe thrombomodulin of this embodiment, similar sugar chains may be addedto similar positions. Two types of N-linked sugar chains, those offucosyl biantennary type and fucosyl triantennary type, may bind to thesoluble thrombomodulin of this embodiment, and ratio thereof is, forexample, 100:0 to 60:40, preferably 95:5 to 60:40, more preferably 90:10to 70:30. The ratio of these sugar chains can be measured on atwo-dimensional sugar chain map described in Biochemical ExperimentalMethods, Vol. 23, Methods of Researches on Glycoprotein Sugar Chains,Japan Scientific Societies Press (1990), and the like. Furthermore, whena sugar composition of the soluble thrombomodulin of this embodiment isexamined, neutral saccharides, aminosaccharides, and sialic acid isdetected, of which content may be, each independently for example, 1 to30%, preferably 2 to 20%, more preferably 5 to 10% in terms of weightratio based on a protein content. The sugar contents can be measured bythe methods described in Lecture of New Biochemical Experiments, Vol. 3,Sugar I, Glycoprotein (Book 1), Tokyo Kagaku Dojin (1990) (neutralsaccharides: phenol-sulfuric acid method, aminosaccharides: Elson-Morganmethod, sialic acid: periodic acid-resorcinol method).

As a signal sequence that can be used for expression where the solublethrombomodulin is obtained by gene manipulation, a nucleotide sequenceencoding the amino acid sequence of the positions 1 to 18 in SEQ ID NO:9, and a nucleotide sequence encoded by a nucleotide sequence encodingthe amino acid sequence of the positions 1 to 16 in SEQ ID NO: 9 can beused, and other known signal sequences such as the signal sequence ofhuman tissue plasminogen activator can also be used (InternationalPublication WO88/9811).

When a DNA sequence encoding soluble thrombomodulin is introduced into ahost cell, there is preferably used a method of incorporating the DNAsequence encoding soluble thrombomodulin into a vector, more preferablyan expression vector that can be expressed in animal cells, and thenintroducing the vector into the host cell. The term “expression vector”means a DNA molecule constituted by a promoter sequence, a sequence foradding a ribosome binding site to mRNA, a DNA sequence encoding aprotein to be expressed, a splicing signal, a terminator sequence fortranscription termination, a replication origin sequence, and the like.Examples of a preferred animal cell expression vector include pSV2-Xreported by R. C. Mulligan et al. (Proc. Natl. Acad. Sci. U.S.A. 78,2072 (1981)) and pBP69T (69-6) reported by P. M. Howley et al. (Methodsin Emzymology, 101, 387, Academic Press (1983)). Further, there is alsoanother preferred embodiment in which DNA is introduced into anexpression vector expressible in a microorganism.

Examples of host cell that can be used in production of such peptides asmentioned above include animal cells.

Examples of the animal cells include Chinese hamster ovary (CHO) cells,COS-1 cells, COS-7 cells, VERO (ATCC CCL-81) cells, BHK cells, caninekidney-derived MDCK cells, hamster AV-12-664 cells, NS0 cells, and thelike. In addition, examples of host cell derived from human include HeLacells, WI38 cells, human 293 cells, and PER.C6 cells. Of these cells,CHO cells are very common and preferred, and among the CHO cells,dihydrofolate reductase (DHFR)-deficient CHO cells are more preferred.

In a gene manipulation process or a peptide production process,microorganisms such as Escherichia coli are also often used. Ahost-vector system suitable for each process is preferably used, and anappropriate vector system can also be selected for the aforementionedhost cells. A thrombomodulin gene used in a genetic recombinationtechnique has been cloned. Examples of production of thrombomodulin bysuch a gene recombination technique have been disclosed, and further,methods for purifying thrombomodulin to obtain a purified productthereof are also known (Japanese Patent Unexamined Publication Nos.64-6219, 2-255699, 5-213998, 5-310787, 7-155176; and J. Biol. Chem.,264:10351-10353 (1989)). Therefore, the soluble thrombomodulin used forthis embodiment can be produced by using the methods described in theaforementioned reports, or by similar methods. For example, JapanesePatent Unexamined Publication No. 64-6219 discloses the Escherichia coliK-12 strain DH5 (ATCC Accession No. 67283) containing a plasmid pSV2TMJ2that contains a DNA encoding the full-length thrombomodulin. This strainre-deposited at the former National Institute of Bioscience andHuman-Technology (currently Independent Administrative Institution,National Institute of Advanced Industrial Science and Technology,International Patent Organism Depositary) (Escherichia coliDH5/pSV2TMJ2) (FERM BP-5570) can also be used. The thrombomodulin ofthis embodiment can be prepared by a known gene manipulation techniqueusing a DNA encoding the full-length thrombomodulin as a startingmaterial.

The soluble thrombomodulin may be prepared by a conventionally knownmethod or a similar method. For example, the aforementioned method ofYamamoto et al. (Japanese Patent Unexamined Publication No. 64-6219) orthe method described in Japanese Patent Unexamined Publication No.5-213998 can be referred to. Specifically, for example, a DNA encodingthe amino acid sequence of SEQ ID NO: 9 is prepared from a human-derivedsoluble thrombomodulin gene by a gene manipulation technique, and may befurther modified as required. For such modification, in order to obtaina DNA encoding the amino acid sequence of SEQ ID NO: 11 (whichspecifically consists of the nucleotide sequence of SEQ ID NO: 12),codons encoding the amino acid at the position 473 in the amino acidsequence of SEQ ID NO: 9 (in particular, the nucleotide at the position1418) are mutated by site-directed mutagenesis according to the methoddescribed in Methods in Enzymology, 100: 468 (1983), Academic Press. Forexample, by using a synthetic DNA for mutation having the nucleotidesequence of SEQ ID NO: 13, the nucleotide T at the position 1418 in SEQID NO: 10 may be converted to the nucleotide C to obtain a mutated DNA.

The DNA prepared as described above is incorporated into, for example,Chinese hamster ovary (CHO) cells to obtain transformant cells. Suchcells are subjected to appropriate selection, and soluble thrombomodulinpurified by a known method can be produced from a culture solutionobtained by culturing a selected cell. As described above, the DNA (SEQID NO: 10) encoding the amino acid sequence of SEQ ID NO: 9 ispreferably transfected into the aforementioned host cell.

For the culture of the aforementioned transformant cell, a medium usedfor ordinary cell culture may be used, and it is preferable to culturethe transformant cell in various kinds of media in advance to choose anoptimal medium. For example, a known medium such as MEM medium, DMEMmedium, and 199 medium may be used as a base medium, and a furtherimproved medium or a medium added with supplements for various media maybe used. Examples of the culture method include serum culture, in whichculture is performed in a medium containing blood serum, and serum-freeculture, in which culture is performed in a medium not containing bloodserum. Although the culture method is not particularly limited, theserum-free culture is preferred.

When serum is added to a medium in the case of the serum culture, bovineserum is preferred. Examples of bovine serum include fetal bovine serum,neonate bovine serum, calf bovine serum, adult bovine serum, and thelike, and any of these examples may be used so far that the serum issuitable for the cell culture. As the serum-free medium used in theserum-free culture, commercially available media can be used. Serum-freemedia suitable for various cells are marketed, and for example, for theCHO cell, CD-CHO, CHO-S-SFMII and CHO-III-PFM are sold by Invitrogen,and IS CHO, IS CHO-CD medium, and the like are sold by IrvineScientific. These media may be used without any treatment, or they maybe improved or added with supplements and used. Examples of theserum-free medium further include the DMEM medium containing 5 mg/L eachof insulin, transferrin, and selenious acid. As described above, themedium is not particularly limited so far that the medium can be used toproduce the thrombomodulin of this embodiment. The culture method is notparticularly limited, and any of batch culture, repetitive batchculture, fed-batch culture, perfusion culture, and the like may be used.

When the soluble thrombomodulin is prepared by the aforementioned cellculture method, diversity may be observed in the N-terminus amino aciddue to posttranslational modification of the protein. For example, theamino acid of the position 17, 18, 19 or 22 in SEQ ID NO: 9 may serve asthe N-terminus amino acid. Further, for example, the N-terminus aminoacid may be modified so that the glutamic acid at the position 22 ischanged to pyroglutamic acid. It is preferred that the amino acid of theposition 17 or 19 serves as the N-terminus amino acid, and it is morepreferred that the amino acid of the position 19 serves as theN-terminus amino acid. Further, there is also another embodiment inwhich the amino acid of the position 17 serves as the N-terminus aminoacid, which is a preferred embodiment. As for the modification,diversity and the like mentioned above, similar examples can bementioned for the sequence of SEQ ID NO: 11.

Further, when the soluble thrombomodulin is prepared by using a DNAhaving the nucleotide sequence of SEQ ID NO: 10, diversity of theC-terminus amino acid may be observed, and a peptide shorter by oneamino acid residue may be produced. Specifically, the C-terminus aminoacid may be modified so that the amino acid of the position 515 servesas the C-terminus amino acid, and further the position 515 is amidated.Further, a peptide shorter by two amino acid residues may be produced.Specifically, the amino acid of the position 514 may serve as theC-terminus amino acid. Therefore, any of peptides having significantdiversity of the N-terminus amino acid and C-terminus amino acid, or amixture of them may be produced. It is preferred that the amino acid ofthe position 515 or the amino acid of the position 516 serves as theC-terminus amino acid, and it is more preferred that the amino acid ofthe position 516 serves as the C-terminus amino acid. Further, there isalso another embodiment in which the amino acid of the position 514serves as the C-terminus amino acid, which is a preferred embodiment.Concerning the modification, diversity and the like described above, thesame shall apply to a DNA having the nucleotide sequence of SEQ ID NO:12.

The thrombomodulin obtained by the method described above may be amixture of peptides having diversity in the N-terminus and C-terminusamino acids. Specific examples include a mixture of peptides having thesequences of the positions 19 to 516, positions 19 to 515, positions 19to 514, positions 17 to 516, positions 17 to 515, and positions 17 to514 in SEQ ID NO: 9.

Highly-purified soluble thrombomodulin in which contamination of HCP isreduced is provided by the present invention.

Examples of the highly-purified soluble thrombomodulin of thisembodiment include highly-purified soluble thrombomodulin that does notsubstantially contain any protein other than soluble thrombomodulin.Specifically, an example includes a soluble thrombomodulin notsubstantially containing HCP, for example. Preferably, an exampleincludes a soluble thrombomodulin not substantially containing HCP,mouse IgG, and bovine serum proteins.

The highly-purified soluble thrombomodulin of this embodiment containsno proteins originated from human.

The content of HCP is not particularly limited so far that the solublethrombomodulin is in a state that it does not substantially contain HCP.The content is preferably a ratio of HCP being less than 10 ng, morepreferably less than 8 ng, still more preferably less than 7 ng, furthermore preferably less than 6 ng, most preferably less than 5 ng per10,000 U of soluble thrombomodulin. The highly-purified solublethrombomodulin of this embodiment is produced in a transformant cellobtained by transfecting a host cell with a DNA containing a nucleotidesequence encoding soluble thrombomodulin, and it is considered that eventhe product is purified as highly as possible, there still actually is apossibility of contamination of HCP in a trace amount and the like. Acontent of HCP as low as possible is preferred. An example of minimumcontent of HCP includes, for example, a ratio of 0.001 ng of HCP per10,000 U of soluble thrombomodulin.

The content of mouse IgG is not particularly limited so far that thesoluble thrombomodulin is in a state that it does not substantiallycontain mouse IgG. A ratio of less than 10 ng of mouse IgG is preferred,a ratio of less than 2 ng is more preferred, and the ratio of less than0.6 ng is still more preferred per 10,000 U of soluble thrombomodulin.

The content of bovine serum proteins is not particularly limited so farthat the soluble thrombomodulin is in a state that it does notsubstantially contain bovine serum proteins. A ratio of less than 10 ngof bovine serum proteins is preferred, a ratio of less than 2 ng is morepreferred, and a ratio of less than 0.6 ng is still more preferred per10,000 U of soluble thrombomodulin. Concentrations of these proteins arepreferably measured by ELISA, and the measurement can be performed byreferring to Biochemical Experimental Methods, Vol. 11, EnzymeImmunoassay, Tokyo Kagaku Dojin (1992), and the like.

As for thrombomodulin purity of soluble thrombomodulin based on thetotal proteins in the highly-purified soluble thrombomodulin of thisembodiment, the purity is preferably 99% or higher, more preferably99.5% or higher, still more preferably 99.7% or higher, most preferably99.9% or higher according to HPLC method. A type of the chromatographyused in the HPLC method is not limited so far that purity of the solublethrombomodulin can be measured. Examples include gel filtration liquidchromatography, ion exchange liquid chromatography, reverse phase liquidchromatography, and the like, and gel filtration liquid chromatographyis preferred. When gel filtration liquid chromatography is used, thecolumn to be used may be chosen depending on the molecular weight of thesoluble thrombomodulin. An example includes, for example, a method ofdevelopment by using a phosphate buffer of pH 7.3 using TOSOH TSKgelG3000SWXL (TOSOH, Japan). The test may be performed according to thedescription in Japanese Pharmacopoeia, Liquid Chromatography <2.01>.

In the highly-purified soluble thrombomodulin of this embodiment, DNAcomponents originated from the host is preferably at a ratio of lessthan 2 ng, more preferably less than 0.2 ng, still more preferably lessthan 0.02 ng per 10,000 U of soluble thrombomodulin. Amount of DNAs canbe easily measured by using Threshold System (Molecular Devices,U.S.A.).

A form of the highly-purified soluble thrombomodulin of this embodimentis not particularly limited so far that the content of HCP is within aratio of less than 10 ng per 10,000 U of soluble thrombomodulin, and itcan exist in the form of a solution or powder. The product preferablyexists in the form of a solution. There is also another embodiment inwhich the product exists in the form of powder, which is a preferredembodiment. As for a concentration in the form of a solution, an upperlimit is preferably 100 mg/mL or lower, more preferably 60 mg/mL orlower, still more preferably 30 mg/mL or lower, most preferably 15 mg/mLor lower, and lower limit is preferably 2 mg/mL or higher, morepreferably 4 mg/mL or higher, still more preferably 6 mg/mL or higher,further more preferably 8 mg/mL or higher, most preferably 10 mg/mL orhigher. Further, when the product exists in the form of a powder, apreferred example includes the form of a lyophilized powder. Suchlyophilized product can be obtained by referring to the method describedin WO03/061687.

The highly-purified soluble thrombomodulin of this embodiment can beobtained so as not to substantially contain endotoxins. The endotoxincontent may preferably be less than 1 endotoxin unit (EU), morepreferably less than 0.2 EU, still more preferably less than 0.04 EU,per 10,000 U of soluble thrombomodulin. Amount of endotoxins can bemeasured in accordance with the descriptions in Japanese Pharmacopoeia,General Test Procedures, Endotoxin Test Method <4.01>. Further, thehighly-purified soluble thrombomodulin of this embodiment can beobtained in a state that it does not contain any substances harmful toliving bodies such as TFA and almost in a sterile state, andaccordingly, can be used as a material for pharmaceutical products.

The highly-purified soluble thrombomodulin of this embodiment in whichcontamination of HCP is reduced can be obtained by bringing a solutioncontaining soluble thrombomodulin into contact with nylon and/orpolyethersulfone. It is preferable to use nylon. There is also anotherembodiment in which it is preferable to use polyethersulfone.

Examples of nylon with which a solution containing the solublethrombomodulin of this embodiment is brought into contact include, forexample, polyamides containing an aliphatic structure such as Nylon 6,Nylon 66, Nylon 46, and Nylon MXD 6. The type of nylon is not limited sofar that the nylon can adsorb HCP. Nylon 6 is preferred. Nylon isavailable as, for example, Minisart NY sold by Sartorius. The form ofnylon is not particularly limited so far that the nylon is in such aform that a solution can be contacted, such as those of membrane,nonwoven fabric, and beads. The nylon is preferably molded in the formof membrane and used as a filtration membrane. In this embodiment, apore diameter of the filtration membrane is not limited so far that thediameter allows HCP to pass through the membrane, for example, 0.01 to10 μm, preferably 0.1 to 1 μm, more preferably 0.01 to 0.06 μm. Thevolume of the solution containing the soluble thrombomodulin to becontacted with nylon can be easily determined by bringing a part of thesolution into contact with a small amount of nylon beforehand toevaluate the HCP-removing ability thereof.

The highly-purified soluble thrombomodulin of this embodiment can beprepared with confirming that the HCP content in the solution obtainedby bringing a soluble thrombomodulin-containing solution containing HCPinto contact with nylon is in a ratio of less than 10 ng of HCP per10,000 U of soluble thrombomodulin, and when the HCP content becomes 10ng or higher per 10,000 U of soluble thrombomodulin, collection ofhighly-purified soluble thrombomodulin can be terminated, or after theused nylon is changed to fresh nylon, the collection of highly-purifiedsoluble thrombomodulin may be restarted. As for examples of the relationbetween the amount of HCP and the area of nylon, examples where a nylonis in the form of filtration membrane for example include an upper limitof the area of the membrane being 50 m² or smaller, preferably 5 m² orsmaller, more preferably 0.5 m² or smaller, still more preferably 0.1 m²or smaller, and preferably a lower limit being 0.01 m² or larger, morepreferably 0.02 m² or larger, still more preferably 0.03 m² or larger,per 1 mg of HCP. It is important to determine the membrane areadepending on the amount of HCP desired to be reduced.

The polyethersulfone with which a solution containing the solublethrombomodulin of this embodiment is brought into contact is availableas, for example, Minisart High-Flow sold by Sartorius. The form ofpolyethersulfone is not particularly limited so far that it is in such aform that a solution can be contacted, such as those of membrane,nonwoven fabric, and beads. Polyethersulfone is preferably molded in theform of membrane and used as a filtration membrane. In this embodiment,a pore diameter of the filtration membrane is not limited so far thatthe diameter allows HCP to pass through the membrane, for example, 0.01to 10 μm, preferably 0.1 to 1 μm, more preferably 0.01 to 0.06 μm. Thevolume of the solution containing the soluble thrombomodulin to becontacted with polyethersulfone can be easily determined by bringing apart of the solution into contact with a small amount ofpolyethersulfone beforehand to evaluate the HCP-removing abilitythereof.

The highly-purified soluble thrombomodulin of this embodiment can becollected with confirming that the HCP content in the solution obtainedby bringing a soluble thrombomodulin-containing solution containing HCPinto contact with polyethersulfone is in a ratio of less than 10 ng per10,000 U of soluble thrombomodulin, and when the HCP content becomes 10ng or higher per 10,000 U of soluble thrombomodulin, collection ofhighly-purified soluble thrombomodulin can be terminated, or after theused polyethersulfone is changed to fresh polyethersulfone, thecollection of highly-purified soluble thrombomodulin may be restarted.As for examples of the relation between the amount of HCP and the areaof polyethersulfone, examples where a polyethersulfone is in the form offiltration membrane for example include an upper limit of the area ofthe membrane being 50 m² or smaller, preferably 5 m² or smaller, morepreferably 0.5 m² or smaller, still more preferably 0.1 m² or smaller,and preferably a lower limit being 0.01 m² or larger, more preferably0.02 m² or larger, still more preferably 0.03 m² or larger, per 1 mg ofHCP. It is important to determine the membrane area depending on theamount of HCP desired to be reduced.

The production process of the highly-purified soluble thrombomodulin ofthis embodiment in which contamination of HCP is reduced is notparticularly limited, so far that the process comprises the step ofbringing a solution containing soluble thrombomodulin into contact withnylon and/or polyethersulfone so that the HCP content can be in a ratioof less than 10 ng per 10,000 U of soluble thrombomodulin. An exampleincludes the following production process:

A production process comprising the steps of (a) to (g), and the step ofbringing a solution containing soluble thrombomodulin into contact withnylon and/or polyethersulfone:(a) the step of culturing a transformant cell and collecting culturemedium (production solution),(b) the step of filtering the production solution to obtain a filteredproduction solution,(c) the step of applying the filtered production solution to anionexchange column chromatography to obtain a roughly purified solution,(d) the step of applying the roughly purified solution to affinitycolumn chromatography using a column carrying anti-thrombomodulinmonoclonal antibody to obtain a purified solution 1,(e) the step of applying the purified solution 1 to cation exchangecolumn chromatography to obtain a purified solution 2,(f) the step of applying the concentrated purified solution 2 to gelfiltration column chromatography, and concentrating the eluate to obtaina purified solution 3, and(g) the step of filtering the purified solution 3 with a virus-removingmembrane and a sterile filtration membrane.

In the aforementioned production process, the step of bringing asolution containing soluble thrombomodulin into contact with nylonand/or polyethersulfone may be included in any one of or two or more ofthe steps (b) to (g). It is preferred that the process includes any oneof or two or more of the steps (d) to (g). In order to efficientlyremove HCP, it is extremely preferable to perform the step of bringingthe solution into contact with nylon and/or polyethersulfone after thefinal step of the production process, i.e., the step (g).

Further, in order to completely obviate contamination of bovine serumproteins, it is more preferred that the culture of the transformant cellin the step (a) is performed as serum-free culture.

Examples of the production process of the highly-purified solublethrombomodulin of this embodiment in which contamination of HCP isreduced also include the following production process:

A production process comprising the steps of (a) to (g), and the step ofbringing a solution containing soluble thrombomodulin into contact withnylon and/or polyethersulfone:(a) the step of culturing a transformant cell and collecting culturemedium (production solution),(b) the step of filtering the production solution to obtain a filteredproduction solution,(c) the step of applying the filtered production solution to anionexchange column chromatography to obtain a roughly purified solution 1,(d) the step of applying the roughly purified solution to hydrophobiccolumn chromatography to obtain a roughly purified solution 2,(e) the step of applying the roughly purified solution 2 to affinitycolumn chromatography using a column carrying anti-thrombomodulinmonoclonal antibody to obtain a purified solution 1,(f) the step of applying the concentrated purified solution 1 to gelfiltration column chromatography to obtain a purified solution 2, and(g) the step of filtering the purified solution 2 with a sterilefiltration membrane.

In the aforementioned production process, the step of bringing asolution containing soluble thrombomodulin into contact with nylonand/or polyethersulfone may be included in any one of or two or more ofthe steps (b) to (g). It is preferable that said step is included in anyone of or two or more of the steps (e) to (g). In order to efficientlyremove HCP, it may be preferable to perform the step of bringing thesolution into contact with nylon and/or polyethersulfone after the finalstep of the production process, i.e., the step (g).

Further, in order to completely obviate contamination of bovine serumproteins, it is more preferred that the culture of the transformant cellin the step (a) is performed as serum-free culture.

The highly-purified soluble thrombomodulin of this embodiment can beprepared with confirming that the HCP content in the solution obtainedby bringing a soluble thrombomodulin-containing solution containing HCPinto contact with nylon and/or polyethersulfone is in a ratio of lessthan 10 ng per 10,000 U of soluble thrombomodulin, and when the HCPcontent becomes 10 ng or larger per 10,000 U of soluble thrombomodulin,the preparation of highly-purified soluble thrombomodulin may beterminated, or after the used nylon and/or polyethersulfone is changedto fresh nylon and/or polyethersulfone, the preparation ofhighly-purified soluble thrombomodulin may be restarted. As describedabove, the production process of the highly-purified solublethrombomodulin of this embodiment in which contamination of HCP isreduced is not particularly limited so far that the process comprisesthe step of bringing a solution containing soluble thrombomodulin intocontact with nylon and/or polyethersulfone, and HCP content becomes in aratio of less than 10 ng per 10,000 U of soluble thrombomodulin. Morespecifically, a purification step may be performed after the step ofbringing the solution into contact with nylon and/or polyethersulfone,and as a result, it is sufficient that highly-purified solublethrombomodulin having an HCP content being in a ratio of less than 10 ngof HCP per 10,000 U of soluble thrombomodulin can be obtained.

Examples of the purification step that may be performed after the stepof bringing the solution into contact with nylon and/or polyethersulfoneinclude steps of performing column chromatography such as anion exchangecolumn chromatography, affinity column chromatography, cation exchangecolumn chromatography, gel filtration column chromatography, andhydrophobic column chromatography, membrane filtration such as membraneconcentration, virus removing, and sterile filtration, or a combinationof two or more of these treatments. A step of performing cation exchangecolumn chromatography, gel filtration column chromatography, membraneconcentration, virus removing, sterile filtration, or a combination oftwo or more of these treatments is preferred. It may be preferable toperform cation exchange column chromatography, gel filtration columnchromatography, membrane concentration, virus removing, and sterilefiltration after the step of bringing the solution into contact withnylon and/or polyethersulfone. As a more preferred example, thepurification step comprising cation exchange column chromatography, gelfiltration column chromatography, membrane concentration, virusremoving, and sterile filtration may be performed by performing cationexchange column chromatography, membrane concentration, gel filtrationcolumn chromatography, membrane concentration, virus removing, andsterile filtration in this order, after the step of bringing thesolution into contact with nylon and/or polyethersulfone. Examples ofthe material used for the cation exchange column chromatography includeSP Sepharose Fast Flow, DEAE Sepharose Fast Flow, Capto S, Capto DEAE(GE Healthcare), S HyperCel (Pall), and TOYOPEARL GigaCap S-650 (TOSOH),and SP Sepharose Fast Flow is preferred. Examples of the concentrationmembrane include MICROZA UF (Asahi Kasei Chemicals), Kvick Flow 10KD (GEHealthcare), and Pellicon 2 (Millipore), and MICROZA UF is preferred.Examples of the material used for the gel filtration columnchromatography include Sephacryl S-300 HR, Superose 12 pg (GEHealthcare), and TOYOPEARL HW (TOSOH), and Sephacryl S-300 HR ispreferred. Examples of the virus-removing membrane include PLANOVA 15N(Asahi Kasei Medical), Biresolve NFP (Millipore), and Ultipor VF (Pall),and PLANOVA 15N is preferred. Examples of the sterile filtrationmembrane include Millipak, Millidisk (Millipore), Supor EVA (Pall), andSartopore 2 (Sartorius Stedim), and Millipak is preferred.

As described above, the highly-purified soluble thrombomodulin of thisembodiment obtained by bringing a solution containing solublethrombomodulin into contact with nylon and/or polyethersulfone containsHCP being in a ratio of less than 10 ng of HCP per 10,000 U of solublethrombomodulin.

One U of the soluble thrombomodulin of this embodiment is defined as anamount that can generate 0.1 μmol of p-nitroaniline per 1 minute in theAPC assay using activation of Protein C as an index, and can be measuredby the method comprising the following steps according to the methoddescribed in Biologicals, 30, 69-76 (2002):

(a) the step of adding human thrombin to a test solution containingsoluble thrombomodulin, and warming the mixture,(b) the step of adding human Protein C, and warming the mixture,(c) the step of adding heparin-antithrombin III, and warming themixture,(d) the step of adding a synthetic substrate S-2366(pyroGlu-Pro-Arg-pNA.HCl), and warming the mixture,(e) the step of adding acetic acid to terminate the substrate cleavingreaction,(f) the step of measuring absorbance at 405 nm, and(g) the step of determining activity of the solublethrombomodulin-containing test solution in accordance with the followingequation:

Activity (U/mL)=[(A _(sample) ×A _(blank))×V ₁]/(M×T×k×V ₂)×Dilutiontime of sample  [Equation 1]

A_(sample): Absorbance of sample solutionA_(blank): Absorbance of blank (water)M: Molar absorption coefficient of p-nitroaniline: 9.6×10⁻³ [1/μM]V₁: Volume at the time of spectrometry (L)V₂: Volume of sample solution (mL)T: Substrate cleaving reaction time (minute)k: Molar number of p-nitroaniline released by activated Protein Cgenerated by 1 U of thrombomodulin: 0.1 (μmol/minute/U)

The highly-purified soluble thrombomodulin of this embodiment has theactivity of, for example, 3,000 U, preferably 4,000 to 9,000 U, morepreferably 5,000 to 8,000 U, still more preferably 6,000 to 7,000 U, per1 mg of the protein. Concentration of the protein can be measured inaccordance with a known method for measuring protein concentration byusing bovine serum albumin as a standard sample. Examples of the methodinclude, for example, Lowry method, Bradford method, BCA method, and thelike.

The HCP content of the highly-purified soluble thrombomodulin of thisembodiment is measured by a method comprising at least the followingsteps:

(a) the step of preparing host cell-originated proteins from culturesupernatant obtained by carrying out serum-free culture of atransformant cell obtained by transfecting a host cell with a DNAcontaining a nucleotide sequence encoding soluble thrombomodulin, or thehost cell,(b) the step of purifying an anti-host cell-originated protein antibodyfrom antiserum obtained by sensitizing a rabbit with the hostcell-originated proteins obtained in (a) mentioned above, the step ofconstructing a measurement system comprising:(c1) the step of adsorbing the anti-host cell-originated proteinantibody obtained in (b) mentioned above to a solid phase,(c2-1) the step of bringing a soluble thrombomodulin-containing testsolution suspected to be contaminated with the host cell-originatedproteins into contact with the solid phase to which the anti-hostcell-originated protein antibody is adsorbed,(c3) the step of adding a biotinylated anti-host cell-originated proteinantibody to the solid phase,(c4) the step of adding a solution of avidinylated peroxidase to thesolid phase,(c5) the step of adding an enzyme substrate solution to allow colordevelopment, and(c6) the step of terminating the color development and measuringabsorbance,(d) the step of measuring concentration of the host cell-originatedproteins in the soluble thrombomodulin-containing test solutionsuspected to be contaminated with the host cell-originated proteins inthe aforementioned measurement system, and determining whether theconcentration of the host cell-originated proteins in the solublethrombomodulin-containing test solution is within a range that enablesquantification of the proteins in the aforementioned measurement system,which range is confirmed beforehand by performing measurement using asolution of the host cell-originated proteins of a known concentrationin the aforementioned measurement system,(e-1) the step of determining the concentration of the hostcell-originated proteins in the soluble thrombomodulin-containing testsolution suspected to be contaminated with the host cell-originatedproteins as the concentration of the host cell-originated proteins inthe solution, when the concentration is determined to be within therange that enables the quantification in (d) mentioned above,(e-2-1) the step of concentrating or diluting the solublethrombomodulin-containing test solution suspected to be contaminatedwith the host cell-originated proteins, if desired, to make theconcentration of the host cell-originated proteins to be a measurableconcentration within the range that enables the quantification in theaforementioned measurement system, when the concentration of the hostcell-originated proteins is determined to be not within the range thatenables the quantification in (d) mentioned above, and recording theconcentration ratio or dilution ratio,(e-2-2) the step of measuring the host cell-originated proteinconcentration in the soluble thrombomodulin-containing test solutionconcentrated or diluted in (e-2-1) mentioned above in a measurementsystem corresponding to the measurement system represented by the stepsof (c1) to (c6) mentioned above in which (c2-1) is replaced with(c2-2) mentioned below, and obtaining the host cell-originated proteinconcentration with taking the concentration ratio or dilution ratio intoconsideration,(c2-2) the step of bringing the soluble thrombomodulin-containing testsolution concentrated or diluted, if necessary, into contact with thesolid phase to which the anti-host cell-originated protein antibody isadsorbed, and the step of bringing a solution containing the hostcell-originated proteins of a known concentration into contact with thesolid phase to which the anti-host cell-originated protein antibody isadsorbed, and(f) the step of calculating ratio of the host cell-originated proteinconcentration obtained in (e-1) or (e-2-2) based on APC activity ofthrombomodulin per unit volume of the soluble thrombomodulin-containingtest solution measured separately.

In this specification, HCP means proteins originated from the host cellsused for preparing gene recombinant cells that produce solublethrombomodulin, and does not mean to include soluble thrombomodulin. HCPcan be prepared from culture supernatant obtained by culturing hostcells of the same type as those of the cells used for the transfectionwith a DNA containing the nucleotide sequence encoding thrombomodulin.In the case of CHO cell, for example, the term of host cell of the sametype means a concept encompassing cells of strains classified into CHOcells, such as those of the cell lines CHO-K1 (ATCC No. CCL-61), CHO-S(Invitrogen, U.S.A., Catalog No. 11619-012), CHO-DXB11, and CHO-DG44(Invitrogen, U.S.A., Catalog No. 12610-010), and the preparation may beperformed by using any of cell lines classified into CHO cells. As forthe CHO cell, it is preferable to use cells of the cell line DXB11 orCHO-K1, more preferably cells of the cell line DXB11, as the host cellof the same type. There is also another embodiment in which it ispreferable to use cells of the cell line CHO-K1.

HCP means proteins originated from the host cells used for preparing thegene recombinant cells that produce soluble thrombomodulin, and isdefined to be measurable by the method including at least theaforementioned steps (a) to (f). Examples of constituents of HCPinclude, as shown in Test Example 6, histone H2B (Biochimie, 61 (1),61-69 (1979)).

Further, when preparation is carried out from the culture supernatantobtained by culturing the transformant cell obtained by transfecting ahost cell of the same type with a DNA containing a nucleotide sequenceencoding thrombomodulin, the culture supernatant can be applied to anantibody column using an antibody that specifically binds tothrombomodulin as the ligand, and a non-adsorbed fraction can becollected. After it is confirmed that the APC activity of thrombomodulinis not detected in this non-adsorbed fraction, the fraction can be usedas HCP. HCP is preferably concentrated by using an ultrafiltrationmembrane, as required. In addition, in order to avoid contamination ofother proteins, the medium used for culturing the host cell or thetransformant cell is preferably a serum-free medium, and it is morepreferred that the serum-free medium is a protein-free medium. For thepurification of the anti-HCP antibody from an anti-HCP antiserumobtained by sensitizing a rabbit with HCP, column chromatography can beused, and for example, a combination of ammonium sulfate salting-out andcolumn chromatography can be used. For the column chromatography for thepurification of the anti-HCP antibody, it is preferable to use a ProteinA column. There is also another embodiment in which, when a transformantcell obtained by transfecting a host cell with a DNA containing anucleotide sequence encoding thrombomodulin is used for the preparationof HCP, it is preferred that a thrombomodulin column is used forpurification of the anti-HCP antibody after the purification with aProtein A column, and the non-adsorbed fraction is collected and used asthe anti-HCP antibody.

When an HCP concentration measurement system is constructed, it isnecessary to clarify the quantifiable range thereof, and thequantifiable range is not limited so far that an HCP content of lessthan 10 ng of HCP per 10,000 U of thrombomodulin can be measured. It ismore preferable that a lower concentration can be measured. Thequantifiable range is defined to be, for example, 100 ng/mL or higher,preferably 50 ng/mL or higher, more preferably 25 ng/mL or higher, andfor example, 500 ng/mL or lower.

When the test solution is concentrated, it can be concentrated by ausual protein concentration method, and the method is not particularlylimited. However, it is preferably concentrated with an ultrafiltrationmembrane. Further, there is also another embodiment in which it ispreferable to concentrate the test solution by lyophilizing thesolution, and then dissolving the product with a small volume of wateror buffer. Components other than HCP are also concentrated by theconcentration and may affect the HCP measurement system. Accordingly, itis necessary to concentrate the test solution in such a degree that theHCP measurement system is not affected. For example, upper limit of theconcentration of the soluble thrombomodulin-containing test solution notaffecting the HCP measurement system is, for example, 5 mg/mL.

The HCP content per 10,000 U of thrombomodulin is calculated inaccordance with the following equation.

a/b×10,000

a: HCP content per 1 mL of sample (ng/mL)b: APC activity of thrombomodulin per 1 mL of sample (U/mL)

The highly-purified soluble thrombomodulin of this embodiment promotesthe activation of Protein C by thrombin to provide generation of a largeamount of active Protein C that has an anti-blood coagulation action anda thrombolysis action. Therefore, the highly-purified solublethrombomodulin of this embodiment greatly contributes to anti-bloodcoagulation and thrombolysis in a living body. The highly-purifiedsoluble thrombomodulin of this embodiment has an anti-blood coagulationaction, a platelet aggregation inhibition action, and a thrombolysisaction. Accordingly, the product can be used for a pharmaceuticalcomposition for controlling blood coagulation, or controlling plateletaggregation. Specifically, it can be used for prophylactic andtherapeutic treatments of diseases including, for example, myocardialinfarction, thrombosis, embolism, peripheral vessel obstruction,obstructive arteriosclerosis, disseminated intravascular coagulation(DIC), angina pectoris, transient cerebral ischemic attack, toxemia ofpregnancy, and the like.

When the pharmaceutical composition of this embodiment is prepared, thehighly-purified soluble thrombomodulin of this embodiment and apharmaceutically acceptable carrier can be mixed. More specifically, apharmaceutical composition suitable for effective administration topatients can be prepared by mixing the highly-purified solublethrombomodulin of this embodiment in an amount effective for aprophylactic or therapeutic treatment of any of the diseases mentionedabove with an appropriate amount of carrier. As the pharmaceuticalcomposition of this embodiment, a lyophilized preparation can bepreferably prepared. Further, the pharmaceutical composition of thisembodiment is preferably used as a preparation for intravascularinjection. The composition can also be preferably prepared as apreparation for intravenous infusion. A lyophilized preparation can beprepared by referring to the method described in WO03/061687.

When the composition is used as an injection, the aforementioned carrieris preferably a carrier that can be administered as a pharmaceutical,and can be dissolved in physiological saline or glucose injection.Examples of the carrier include one or more selected from the groupconsisting of sucrose, purified gelatin, albumin, mannitol, glucose, andsodium chloride. For example, a pH adjustor consisting of any of variousmineral salts, and the like are also preferably added. In such a case,the whole pharmaceutical composition as a combination with thehighly-purified soluble thrombomodulin of this embodiment is soluble,and can be finely lyophilized, and thus such a composition is preferred.Further, in this embodiment, it is also preferred that theaforementioned carrier is glycerol. The aforementioned carrier ispreferably added at the time of preparing the composition. The carriertmay also be added when the composition is dissolved before use.

A dose of the highly-purified soluble thrombomodulin of this embodimentfor one time of administration to an adult may change depending on age,sex, body weight, symptoms, and the like. The doses may generally beabout 0.1 to 200 mg, and may be administered, for example, once orseveral times, as required, per day by intravascular injection,preferably intravenous drip infusion. The pharmaceutical composition ofthis embodiment may also be administered so that a dose can be 0.1 to200 mg of the soluble thrombomodulin as the active ingredient, forexample, once or several times, as required, per day by intravascularinjection, preferably intravenous drip infusion.

EXAMPLES

The present invention will be more specifically explained with referenceto the following examples. However, the scope of the present inventionis not limited at all by these.

Reference Example 1 Method for Measuring APC Activity of Thrombomodulin

According to the description of Biologicals, 30, 69-76 (2002), the APCactivity of thrombomodulin is measured on the basis of activation ofProtein C as an index.

A 20 mM calcium chloride solution (75 μL) is added with 25 μL of asample solution diluted with a Tris-imidazole buffer containing 0.05%polysorbate 20, the mixture is cooled on ice, and then added with 25 μLof a 40 U/mL solution of human thrombin (Sigma, U.S.A.), and the mixtureis stirred and warmed at 37° C. Ten minutes after the addition of thehuman thrombin solution, the mixture was added with 25 μL of a 12 U/mLsolution of human Protein C (Enzyme Research, U.S.A.), and the mixturewas stirred and warmed at 37° C. Ten minutes after the addition of thehuman Protein C solution, the mixture was added with 100 μL of aheparin-antithrombin III solution, and the mixture was stirred andwarmed at 37° C. Ten minutes after the addition of theheparin-antithrombin III solution, the mixture was added with 250 μL ofa synthetic substrate S-2366 (ChromoGenics, Sweden) solution warmed at37° C. beforehand, and the mixture is stirred and warmed at 37° C. Tenminutes after the addition of the substrate solution, the mixture wasadded with 1.5 mL of 50% acetic acid, the mixture is stirred, andabsorbance of the mixture is measured at 405 nm by using water as ablank.

The APC activity of thrombomodulin is calculated in accordance with thefollowing equation. One U of thrombomodulin is defined as an amount thatcan generate 0.1 μmol of p-nitroaniline per 1 minute.

Activity (U/mL)=[(A _(sample) −A _(bank))×V ₁]/(M×T×k×V ₂)×Dilution timeof sample  [Equation 2]

A_(sample): Absorbance of sample solutionA_(blank): Absorbance of blank (water)M: Molar absorption coefficient of p-nitroaniline: 9.6×10⁻³ [1/μM]V₁: Volume at the time of spectrometry: 2.0×10⁻³ (L)V₂: Volume of sample solution: 0.025 (mL)T: Substrate cleaving reaction time: 10 (minute)k: Molar number of p-nitroaniline released by activated Protein Cgenerated with 1 U of thrombomodulin: 0.1 (μmol/minute/U)

The reagents are as follows.

<Tris-Imidazole Buffer>

Solution B (100 mL) is added with Solution A, and the mixture isadjusted to pH 8.4, and diluted 10 times with water.

Solution A: 2-Amino-2-hydroxymethyl-1,3-propanediol (3.03 g) andimidazole (1.70 g) are dissolved in 1 M hydrochloric acid (50 mL), thesolution was added with water to a volume of 100 mL, and sodium chloride(11.7 g) is added to the solution and dissolved. Solution B:2-Amino-2-hydroxymethyl-1,3-propanediol (4.04 g), imidazole (2.27 g),and sodium chloride (1.95 g) are dissolved in water to obtain a volumeof 100 mL, and sodium chloride (11.7 g) is added to the solution anddissolved.

<20 mM Calcium Chloride Solution>

A 60 mM calcium chloride solution (1 mL) is added with a Tris-imidazolebuffer (2 mL).

<Heparin-Antithrombin III Solution>

An antithrombin III solution (2 U/mL, 7.5 μL, Mitsubishi Pharma, Japan),a Tris-imidazole buffer (42.5 μL), and a 30 U/mL heparin solution (50μL, Mochida Pharmaceutical, Japan) are mixed by shaking. This solutionis prepared before use, and cooled on ice until just before use.

Reference Example 2 Method for Measuring HCP Concentration

Serum-free culture of gene recombinant CHO cells introduced with thethrombomodulin gene is performed. The culture supernatant is applied onan anti-thrombomodulin antibody column to obtain a non-adsorbedfraction. According to the description of Reference Example 1, the APCactivity of thrombomodulin in this non-adsorbed fraction is measured toconfirm that the activity is not detected, then the fraction isconcentrated with an ultrafiltration membrane, and the concentrate isused as HCP. Anti-HCP antiserum obtained by sensitizing a rabbit withHCP as an antigen is purified by ammonium sulfate salting-out and with aProtein A column, and then applied on an affinity column usingthrombomodulin as the ligand to obtain a non-adsorbed fraction. Asdescribed above, a rabbit anti-HCP antibody that does not recognizethrombomodulin is obtained.

A sample solution is obtained by dilution with PBS containing 0.05%polysorbate 80 so that expected HCP concentration becomes 0 to 500ng/mL. When the HCP concentration of a sample solution is low, thesolution is concentrated to an appropriate concentration by using anultrafiltration membrane or the like. Separately, HCP is added with PBScontaining 0.05% polysorbate 80 to prepare eight kinds of solutionscontaining 500, 400, 300, 200, 100, 50, 25, and 0 ng of HCP in 1 mL asstandard solutions.

A 25 μg/mL rabbit anti-HCP antibody solution diluted with a sodiumcarbonate buffer is added to a 96-well polystyrene plate in a volume of100 μL per well, and the plate is left standing at 25° C. for about 2hours. Then, each well is washed 5 times with 250 μL of PBS containing0.05% polysorbate 80, PBS containing 1% gelatin (200 μL) is added toeach well, and the plate is left standing at 25° C. for about 1 hour.Each well is washed 5 times with 250 μL of PBS containing 0.05%polysorbate 80, then a sample solution and the standard solutions (100μL) are added to the wells, and the plate is left standing at 25° C. forabout 16 hours. Then, each well is washed 5 times with 250 μL of PBScontaining 0.05% polysorbate 80, then a biotinylated rabbit anti-HCPantibody solution (100 μL) is added to each well, and the plate is leftstanding at 25° C. for about 2 hours. Each well is washed 5 times with250 μL of PBS containing 0.05% polysorbate 80, then an avidin-peroxidasesolution (100 μL) is added to each well, and the plate is left standingat 25° C. for about 2 hours. Each well is washed 5 times with 250 μL ofPBS containing 0.05% polysorbate 80, then an enzyme substrate solution(100 μL) is added to each well, and the plate is left standing at roomtemperature in a dark place. When a color is appropriately developed, 50μL of 25% sulfuric acid is added to each well to terminate the reaction,and absorbance of the mixture is measured at 492 nm with anabsorptiometer for 96-well plates (Tecan Japan, Japan). By using acalibration curve prepared with the standard solutions, HCP content inthe sample (1 mL) is calculated. The measurement limit of thismeasurement method is, for example, 25 ng/mL. In accordance with thefollowing equation, HCP content per 10,000 U of thrombomodulin iscalculated, as required.

HCP content per 10,000 U of thrombomodulin (ng/10,000 U)=a/b×10,000

a: HCP content per 1 mL of sample (ng/mL)b: APC activity of thrombomodulin per 1 mL of sample (U/mL)

The reagents are as follows.

<Sodium Carbonate Buffer>

Anhydrous sodium carbonate (0.16 g), and sodium hydrogencarbonate (0.29g) are added to water and dissolved to obtain a volume of 100 mL.

<Avidin-Peroxidase Solution>

A stock solution of horseradish peroxidase bound with avidin D (VectorLaboratories, U.S.A.) is diluted about 30,000 times with PBS containing0.05% polysorbate 80.

<Enzyme Substrate Solution>

Ortho-phenylenediamine dihydrochloride (10 mg) is added to 20 mL of acitrate/phosphate buffer (citric acid monohydrate (2.56 g) and disodiumhydrogenphosphate dodecahydrate (9.12 g) are dissolved in water toobtain a volume of 500 mL) and dissolved, and aqueous hydrogen peroxide(10 μL) is added immediately before use.

Reference Example 3 Method for Measuring Mouse IgG Concentration

Anti-mouse IgG antiserum obtained by sensitizing a rabbit with mouse IgGas an antigen is purified by ammonium sulfate salting-out and with aProtein A column to obtain a rabbit anti-mouse IgG antibody.

A sample solution is obtained by dilution with PBS containing 0.05%polysorbate 80 so that expected mouse IgG concentration becomes 0 to 25ng/mL. When the IgG concentration of a sample solution is low, thesolution is concentrated to an appropriate concentration by using anultrafiltration membrane or the like. Separately, mouse IgG is addedwith PBS containing 0.05% polysorbate 80 to prepare eight kinds ofsolutions containing 25, 20, 15, 10, 5, 2.5, 1.25, 0.63, and 0 ng of IgGin 1 mL as standard solutions.

A 1.5 μg/mL rabbit anti-mouse IgG antibody solution diluted with asodium carbonate buffer is added to a 96-well polystyrene plate in avolume of 100 μL per well, and the plate is left standing at 25° C. forabout 2 hours. Then, each well is washed 5 times with 250 μL of PBScontaining 0.05% polysorbate 80, PBS containing 1% gelatin (200 μL) isadded to each well, and the plate is left standing at 25° C. for about 1hour. Each well is washed 5 times with 250 μL of PBS containing 0.05%polysorbate 80, then a sample solution and the standard solutions (100μL) are added to the wells, and the plate is left standing at 25° C. forabout 16 hours. Then, each well is washed 5 times with 250 μL of PBScontaining 0.05% polysorbate 80, and then a biotinylated rabbitanti-mouse IgG antibody solution (100 μL) is added to each well, and theplate is left standing at 25° C. for about 2 hours. Each well is washed5 times with 250 μL of PBS containing 0.05% polysorbate 80, then anavidin-peroxidase solution (100 μL) is added to each well, and the plateis left standing at 25° C. for about 2 hours. Each well is washed 5times with 250 μL of PBS containing 0.05% polysorbate 80, then an enzymesubstrate solution (100 μL) is added to each well, and the plate is leftstanding at room temperature in a dark place. When a color isappropriately developed, 50 μL of 25% sulfuric acid is added to eachwell to terminate the reaction, and absorbance of the mixture ismeasured at 492 nm with an absorptiometer for 96-well plates (TecanJapan, Japan). By using a calibration curve prepared with the standardsolutions, mouse IgG content in the sample (1 mL) is calculated. Themeasurement limit of this measurement method is, for example, 0.63ng/mL. In accordance with the following equation, mouse IgG content per10,000 U of thrombomodulin is calculated, as required.

Mouse IgG content per 10,000 U of thrombomodulin (ng/10,000U)=a/b×10,000

a: Mouse IgG content per 1 mL of sample (ng/mL)b: APC activity of thrombomodulin per 1 mL of sample (U/mL)

The reagents are as follows.

<Sodium Carbonate Buffer>

Anhydrous sodium carbonate (0.16 g), and sodium hydrogencarbonate (0.29g) are added to water and dissolved to obtain a volume of 100 mL.

<Avidin-Peroxidase Solution>

A stock solution of horseradish peroxidase bound with avidin D (VectorLaboratories, U.S.A.) is diluted about 30,000 times with PBS containing0.05% polysorbate 80.

<Enzyme Substrate Solution>

Ortho-phenylenediamine dihydrochloride (10 mg) is added to 20 mL of acitrate/phosphate buffer (citric acid monohydrate (2.56 g) and disodiumhydrogenphosphate dodecahydrate (9.12 g) are dissolved in water toobtain a volume of 500 mL) and dissolved, and aqueous hydrogen peroxide(10 μL) is added immediately before use.

Reference Example 4 Method for Measuring Bovine Serum ProteinConcentration

Anti-bovine serum protein antiserum obtained by sensitizing a rabbitwith bovine serum as an antigen is purified by ammonium sulfatesalting-out and with a Protein A column to obtain a rabbit anti-bovineserum protein antibody.

A sample solution is obtained by dilution with PBS containing 0.05%polysorbate 80 so that expected bovine serum protein concentrationbecomes 0 to 25 ng/mL. When the bovine serum protein concentration of asample solution is low, the solution is concentrated to an appropriateconcentration by using an ultrafiltration membrane or the like.Separately, bovine serum is added with PBS containing 0.05% polysorbate80 to prepare eight kinds of solutions containing 25, 20, 15, 10, 5,2.5, 1.25, and 0 ng of bovine serum proteins in 1 mL as standardsolutions.

A 10 μg/mL rabbit anti-bovine serum protein antibody solution dilutedwith a sodium carbonate buffer is added to a 96-well polystyrene platein a volume of 100 μL per well, and the plate is left standing at 25° C.for about 2 hours. Then, each well is washed 5 times with 250 μL of PBScontaining 0.05% polysorbate 80, PBS containing 1% gelatin (200 μL) isadded to each well, and the plate is left standing at 25° C. for about 1hour. Each well is washed 5 times with 250 μL of PBS containing 0.05%polysorbate 80, then a sample solution and the standard solutions (100μL) are added to the wells, and the plate is left standing at 25° C. forabout 16 hours. Then, each well is washed 5 times with 250 μL of PBScontaining 0.05% polysorbate 80, then a biotinylated rabbit anti-bovineserum protein antibody solution (100 μL) is added to each well, and theplate is left standing at 25° C. for about 2 hours. Each well is washed5 times with 250 μL of PBS containing 0.05% polysorbate 80, then anavidin-peroxidase solution (100 μL) is added to each well, and the plateis left standing at 25° C. for about 2 hours. Each well is washed 5times with 250 μL of PBS containing 0.05% polysorbate 80, then an enzymesubstrate solution (100 μL) is added to each well, and the plate is leftstanding at room temperature in a dark place. When a color isappropriately developed, 50 μL of 25% sulfuric acid is added to eachwell to terminate the reaction, and absorbance of the mixture ismeasured at 492 nm with an absorptiometer for 96-well plates (TecanJapan, Japan). By using a calibration curve prepared with the standardsolutions, bovine serum protein content in the sample (1 mL) iscalculated. The measurement limit of this measurement method is, forexample, 1.25 ng/mL. In accordance with the following equation, bovineserum protein content per 10,000 U of thrombomodulin is calculated, asrequired.

Bovine serum protein content per 10,000 U of thrombomodulin (ng/10,000U)=a/b×10,000

a: Bovine serum protein content per 1 mL of sample (ng/mL)b: APC activity of thrombomodulin per 1 mL of sample (U/mL)

The reagents are as follows.

<Sodium Carbonate Buffer>

Anhydrous sodium carbonate (0.16 g), and sodium hydrogencarbonate (0.29g) are added to water and dissolved to obtain a volume of 100 mL.

<Avidin-Peroxidase Solution>

A stock solution of horseradish peroxidase bound with avidin D (VectorLaboratories, U.S.A.) is diluted about 30,000 times with PBS containing0.05% polysorbate 80.

<Enzyme Substrate Solution>

Ortho-phenylenediamine dihydrochloride (10 mg) is added to 20 mL of acitrate/phosphate buffer (citric acid monohydrate (2.56 g) and disodiumhydrogenphosphate dodecahydrate (9.12 g) are dissolved in water toobtain a volume of 500 mL) and dissolved, and aqueous hydrogen peroxide(10 μL) is added immediately before use.

Comparative Example 1 Preparation of Soluble Thrombomodulin 1

A gene recombinant CHO cell into which a DNA encoding the amino acidsequence of SEQ ID NO: 9 was introduced was prepared by a geneticmanipulation technique according to Japanese Patent UnexaminedPublication No. 11-341990, Example 1, then inoculated into the DMEMmedium (Invitrogen, U.S.A.) containing 150 mg/L of L-proline (Ajinomoto,Japan), 60 mg/L of kanamycin sulfate (Meiji Seika, Japan), 1 mg/L oftylosin tartrate (Mercian, Japan), and 10% bovine serum (HyClone,U.S.A.), and cultured at 37° C. in a CO₂ incubator. The cells obtainedby centrifuging the culture medium were suspended in the DMEM medium(Invitrogen, U.S.A.) containing 150 mg/L of L-proline (Ajinomoto,Japan), 60 mg/L of kanamycin sulfate (Meiji Seika, Japan), 1 mg/L oftylosin tartrate (Mercian, Japan), 10% dimethyl sulfoxide (Merck,Germany), and 10% bovine serum (HyClone, U.S.A.), and the medium wasdispensed into vials (4×10⁷ cells/vial), and cryopreserved in liquidnitrogen.

Cell culture was performed by using, as the base medium, the mediumdescribed in Japanese Patent Unexamined Publication No. 11-341990,Ingredient Table 2, provided that NaHCO₃ concentration was changed to5,700 mg/L, and NaCl concentration was changed to 2,410 mg/L. Growthmedium was obtained by adding 60 mg/L of kanamycin sulfate (Invitrogen,U.S.A.), 1 mg/L of tylosin tartrate (Sigma-Aldrich, U.S.A.), and 8%bovine serum (HyClone, U.S.A.) to the base medium, and used. Further,production medium was the same as the growth medium, provided that theserum concentration was changed to 3%.

The cells of one vial were thawed, inoculated into 100 mL of the growthmedium, and cultured with stirring at 37° C. for 5 days by using aspinner flask in a CO₂ incubator. When the living cell density became7.0×10⁵ cells/mL or more, the entire volume of the culture medium wastransferred to 0.9 L of the growth medium, and the cells were culturedwith stirring at 37° C. for 5 days by using a spinner flask in a CO₂incubator. When the living cell density became 7.0×10⁵ cells/mL or more,the entire volume of the culture medium was transferred to 9 L of thegrowth medium, and the cells were cultured with stirring at 37° C., pH7.2 and 50% of dissolved oxygen for 5 days by using a culture tank. Whenthe living cell density became 7.0×10⁵ cells/mL or more, the entirevolume of the culture medium was transferred to 120 L of the growthmedium, and the cells were cultured with stirring at 37° C., pH 7.2 and50% of dissolved oxygen for 7 days by using a perfusion culture tank.When the living cell density became 7.0×10⁵ cells/mL or more, perfusionculture was started, in which the production medium was continuouslyadded, and the culture supernatant was continuously collected. Theculture conditions consisted of 37° C., pH 7.2, dissolved oxygen: 50%,medium exchange: 130 to 200 L/day, and surface pressurization: 0 to 0.2MPa. After the living cell density reached 7.5×10⁶ cells/mL, the culturewas further continued for 36 days, and the culture supernatant wascollected as a production solution. The collected production solutionwas clarified by using filtration filters, SUPRAdisc II (Pall, U.S.A.)and Supor EBV (Pall, U.S.A.), and stored at 2 to 10° C. as a filteredproduction solution.

About 700 L of the filtered production solution was applied to aQ-Sepharose Fast Flow (GE Healthcare, U.S.A.) column (diameter: 63 cm,height: 25 cm) equilibrated with a 20 mM Tris-hydrochloric acid buffer(pH 7.7) containing 150 mM sodium chloride. Then, the column was washedwith 6 column volumes (CV) of a 20 mM acetate buffer (pH 5.5) containing180 mM sodium chloride, and further washed with a 20 mMTris-hydrochloric acid buffer (pH 7.7) containing 180 mM sodium chlorideuntil absorbance at 280 nm returned to the baseline. Elution was startedwith a 20 mM Tris-hydrochloric acid buffer (pH 7.7) containing 300 mMsodium chloride, and 0.5 column volume of the eluate from the start ofthe peak of absorbance at 280 nm was obtained as a roughly purifiedsolution. The same operation was repeated 6 times to obtain 6 lots ofthe roughly purified solution. The operation was performed at atemperature of 2 to 10° C., and a chromatography flow rate of 109L/hour.

An anti-thrombomodulin monoclonal antibody was prepared by using humanlung-originated thrombomodulin as the antigen according to JapanesePatent Unexamined Publication No. 11-341990, Example 10, contacted andreacted with CNBr-activated Sepharose 4 Fast Flow (GE Healthcare,U.S.A.) to couple the anti-thrombomodulin monoclonal antibody andthereby prepare anti-thrombomodulin monoclonal antibody-bound Sepharose4 Fast Flow, which was filled in a column to obtain a monoclonalantibody column. About 40 L of the roughly purified solution was appliedto the monoclonal antibody column (diameter: 44 cm, height: 13 cm)equilibrated with a 20 mM phosphate buffer (pH 7.3) containing 0.3 Msodium chloride. 6 CV of a 20 mM phosphate buffer (pH 7.3) containing1.0 M sodium chloride was poured into the column, 3 CV of 0.1 M acetatebuffer (pH 5.0) was further poured to wash the column, and elution wasstarted with a 0.1 M glycine-hydrochloric acid buffer (pH 3.0)containing 0.3 M sodium chloride. The eluate corresponding to the startto the end of the peak of absorbance at 280 nm was obtained, and addedwith 1/10 volume of a 0.5 M phosphate buffer (pH 7.3) to obtain apurified solution 1. The same operation was repeated 6 times to obtain 6lots of the purified solution 1. The operation was performed at atemperature of 2 to 10° C., and a chromatography flow rate of 46 L/hour.

About 170 L of the purified solution 1 of the 6 lots was adjusted to pH3.5 with a 1.0 M glycine-hydrochloric acid buffer (pH 2.0), and appliedto an SP-Sepharose FF (GE Healthcare Bioscience, U.S.A.) column(diameter: 45 cm, height: 10 cm) equilibrated with a 0.1 Mglycine-hydrochloric acid buffer (pH 3.5) containing 0.3 M NaCl. Washingwas started with a 0.1 M glycine-hydrochloric acid buffer (pH 3.5)containing 0.3 M NaCl, and a flow-through fraction corresponding to thestart to the end of the peak of absorbance at 280 nm was obtained, andimmediately neutralized to pH 7 with a 0.5 M phosphate buffer (pH 7.3)to obtain a purified solution 2. The operation was performed at atemperature of 2 to 10° C., and a chromatography flow rate of 160L/hour.

About 200 L of the purified solution 2 was concentrated to about 10 L byusing an ultrafiltration membrane, Microza UF Module SIP-2013 (AsahiKasei Chemicals, Japan), and then applied to a Sephacryl S-300 HR (GEHealthcare Bioscience, U.S.A.) column (diameter: 63 cm, height: 94 cm)equilibrated with a 20 mM phosphate buffer (pH 7.3) containing 50 mMsodium chloride. An elution peak with the maximum absorbance at 280 nmwas separated, and concentrated to about 12 L by using anultrafiltration membrane, Microza UF Module SIP-1013 (Asahi KaseiChemicals, Japan), to obtain a purified solution 3. The operation wasperformed at a temperature of 2 to 10° C., and a chromatography flowrate of 6.2 L/hour.

The purified solution 3 was passed through a virus-removing membrane,PLANOVA 15N (membrane area: 1 m², Asahi Kasei Medical, Japan),equilibrated with a 20 mM phosphate buffer (pH 7.3) containing 50 mMsodium chloride at room temperature and a pressure lower than 0.1 MPa,and then further passed through a 0.22-μm PVDF filtration membrane(Millipore, U.S.A.), and the entire volume of the solution wascollected. The result was used as a purified product of solublethrombomodulin.

By performing the same operation, 3 lots (A1, A2, A3) of the purifiedproduct were obtained.

The APC activities of thrombomodulin of A1, A2, and A3 were 69000 U/mL,68000 U/mL, and 72000 U/mL, respectively.

Soluble thrombomodulin concentrations in the solutions of A1, A2, and A3were 10.5 mg/mL, 10.2 mg/mL, and 10.3 mg/mL, respectively.

Comparative Example 2 Preparation of Soluble Thrombomodulin 2

The medium described in Japanese Patent Unexamined Publication No.11-341990, Ingredient Table 2 was used as the base medium. Growth mediumwas obtained by adding 60 mg/L of kanamycin sulfate (Invitrogen,U.S.A.), 1 mg/L of tylosin tartrate (Sigma-Aldrich, U.S.A.), and 8%bovine serum (HyClone, U.S.A.) to the base medium, and used. Further,production medium was the same as the growth medium, provided that theserum concentration was changed to 4%.

The cells of one vial prepared in Comparative Example 1 were thawed,inoculated into 100 mL of the growth medium, and cultured with stirringat 37° C. for 3 days by using a spinner flask in a CO₂ incubator. Whenthe living cell density became 5.0×10⁵ cells/mL or more, the entirevolume of the culture medium was transferred to 400 mL of the growthmedium, and the cells were cultured with stirring at 37° C. for 3 daysby using a spinner flask in a CO₂ incubator. When the living celldensity became 5.0×10⁵ cells/mL or more, the entire volume of theculture medium was transferred to 2 L of the growth medium, and thecells were cultured with stirring at 37° C. for 3 days by using aspherical bottle in a CO₂ incubator. When the living cell density became5.0×10⁵ cells/mL or more, the entire volume of the culture medium wastransferred to 7.5 L of the growth medium, and the cells were culturedwith stirring at 37° C. for 4 days by using a spherical bottle in a CO₂incubator. When the living cell density became 5.0×10⁵ cells/mL or more,perfusion culture was started, in which the production medium wascontinuously added, and the culture supernatant was continuouslycollected. The culture conditions consisted of 37° C., pH 7.2, dissolvedoxygen: 50%, medium exchange: 10 L/day, and surface pressurization: 0 to0.2 MPa. After the living cell density reached 7.5×10⁶ cells/mL, theculture was further continued for 40 days, and the culture supernatantwas collected as a production solution.

The collected production solution was clarified by using filtrationfilters having pore diameters of 0.7 μm and 0.22 μm (Pall, U.S.A.), andstored at 2 to 10° C. as a filtered production solution.

About 400 L of the filtered production solution was applied to aQ-Sepharose Fast Flow (GE Healthcare, U.S.A.) column (diameter: 44 cm,height: 26 cm) equilibrated with a 20 mM Tris-hydrochloric acid buffer(pH 7.4) containing 150 mM sodium chloride. Then, the column was washedwith 6 CV of a 20 mM acetate buffer (pH 5.5) containing 180 mM sodiumchloride, and further washed with a 20 mM Tris-hydrochloric acid buffer(pH 7.4) containing 180 mM sodium chloride until absorbance at 280 nmreturned to the baseline. Elution was started with a 20 mMTris-hydrochloric acid buffer (pH 7.4) containing 300 mM sodiumchloride, and 0.5 column volume of the eluate from the start of the peakof absorbance at 280 nm was obtained as a roughly purified solution. Theoperation was performed at a temperature of 2 to 10° C., and achromatography flow rate of 45 L/hour.

About 20 L of the roughly purified solution was applied to a monoclonalantibody column (diameter: 44 cm, height: 12 cm) equilibrated with a 20mM phosphate buffer (pH 7.3) containing 0.3 M sodium chloride. 6 CV of a20 mM phosphate buffer (pH 7.3) containing 1.0 M sodium chloride waspoured into the column, 3 CV of a 0.1 M acetate buffer (pH 5.0) wasfurther poured to wash the column, and elution was started with a 0.1 Mglycine-hydrochloric acid buffer (pH 3.0) containing 0.3 M sodiumchloride. The eluate corresponding to the start to the end of the peakof absorbance at 280 nm was obtained, and added with 1/10 volume of a0.5 M phosphate buffer (pH 7.3) to obtain a purified solution 1. Theoperation was performed at a temperature of 2 to 10° C., and achromatography flow rate of 45 L/hour.

About 12 L of the purified solution 1 was adjusted to pH 3.5 with a 1.0M glycine-hydrochloric acid buffer (pH 2.0), and applied to anSP-Sepharose FF (GE Healthcare Bioscience, U.S.A.) column (diameter: 14cm, height: 13 cm) equilibrated with a 0.1 M glycine-hydrochloric acidbuffer (pH 3.5) containing 0.3 M NaCl. Washing was started with a 0.1 Mglycine-hydrochloric acid buffer (pH 3.5) containing 0.3 M NaCl, and aflow-through fraction corresponding to the start to the end of the peakof absorbance at 280 nm was obtained, and immediately neutralized to pH7 with a 0.5 M phosphate buffer (pH 7.3) to obtain a purified solution2. The operation was performed at a temperature of 2 to 10° C., and achromatography flow rate of 15 L/hour.

About 16 L of the purified solution 2 was concentrated to about 1.2 L byusing an ultrafiltration membrane, Microza UF Module SIP-1013 (AsahiKasei Chemicals, Japan), and then applied to a Sephacryl S-300 HR (GEHealthcare Bioscience, U.S.A.) column (diameter: 25 cm, height: 85 cm)equilibrated with a 20 mM phosphate buffer (pH 7.3) containing 50 mMsodium chloride. An elution peak with the maximum absorbance at 280 nmwas separated, and concentrated to about 0.8 L by using anultrafiltration membrane, Microza UF Module SIP-1013 (Asahi KaseiChemicals, Japan), to obtain a purified solution 3. The operation wasperformed at a temperature of 2 to 10° C., and a chromatography flowrate of 1 L/hour.

The purified solution 3 was passed through a virus-removing membrane,PLANOVA 15N (membrane area: 0.3 m², Asahi Kasei Medical, Japan),equilibrated with a 20 mM phosphate buffer (pH 7.3) containing 50 mMsodium chloride at room temperature and a pressure lower than 0.1 MPa,and then further passed through a 0.22-μm PVDF filtration membrane(Millipore, U.S.A.), and the entire volume of the solution wascollected. The result was used as a purified product of solublethrombomodulin (lot: B1).

The APC activity of thrombomodulin of B1 was 79000 U/mL.

Soluble thrombomodulin concentration in the solution of B1 was 12.6mg/mL.

Comparative Example 3 Preparation of Soluble Thrombomodulin 3

The DMEM medium (Invitrogen, U.S.A.) was used as the base medium.

Growth medium was obtained by adding 150 mg/L of L-proline (Ajinomoto,Japan), 60 mg/L of kanamycin sulfate (MeijiSeika Pharma, Japan), 1 mg/Lof tylosin tartrate (Mercian, Japan), and 10% bovine serum (HyClone,U.S.A.) to the base medium, and used. Further, production medium was thesame as the growth medium, provided that the serum concentration waschanged to 1 to 3%.

The cells of one vial prepared in Comparative Example 1 were thawed,inoculated into 100 mL of the growth medium, and cultured with stirringat 37° C. for 5 days by using a spinner flask in a CO₂ incubator. Theentire volume of the culture medium was transferred to 400 mL of thegrowth medium, and the cells were cultured with stirring at 37° C. for 5days by using a spinner flask in a CO₂ incubator. The entire volume ofthe culture medium was transferred to 1.6 L of the growth medium, andthe cells were cultured with stirring at 37° C. for 5 days by using aspherical bottle with bubbling air and CO₂ into the medium. The entirevolume of the culture medium was transferred to 6 L of the growthmedium, and the cells were cultured with stirring at 37° C. for 5 daysby using a spherical bottle with bubbling air and CO₂ into the medium.The entire volume of the culture medium was transferred to 56 L of thegrowth medium, and the cells were cultured with stirring at 37° C. for 4days by using a spherical bottle with bubbling air and CO₂ into themedium. After the entire medium was exchanged, the cells were furthercultured for 3 days. Further, after the entire medium was exchanged, andwhen the living cell density reached 1.0×10⁶ cells/mL, the medium waschanged to the production medium. The production solution was collectedevery day by using a continuous centrifugation machine CC-100 (AlfaLaval, Sweden), and the fresh medium was supplemented. The productionculture was carried out for 100 days. The collected production solutionwas clarified by using filtration filters having pore diameters of 0.7μm and 0.22 μm (Pall, U.S.A.), and stored at 2 to 10° C. as a filteredproduction solution.

About 2400 L of the filtered production solution was applied to aQ-Sepharose Fast Flow (GE Healthcare, U.S.A.) column (diameter: 44 cm,height: 25 cm) equilibrated with a 20 mM Tris-hydrochloric acid buffer(pH 7.4) containing 150 mM sodium chloride. Then, the column was washedwith 6 CV of a 20 mM acetate buffer (pH 5.5) containing 180 mM sodiumchloride, and further washed with 2 CV of a 20 mM Tris-hydrochloric acidbuffer (pH 7.4) containing 180 mM sodium chloride. Elution was startedwith a 20 mM Tris-hydrochloric acid buffer (pH 7.4) containing 300 mMsodium chloride, and about 15 L of the eluate from the start of the peakof absorbance at 280 nm was obtained as a roughly purified solution. Theoperation was performed at a temperature of 2 to 10° C., and achromatography flow rate of 45 L/hour.

The aforementioned roughly purified solution 1 was applied to aButyl-Sepharose FF (GE Healthcare Bioscience, U.S.A.) column (diameter:25 cm. height: 10 cm) equilibrated with a 20 mM phosphate buffer (pH7.0) containing 0.3 M NaCl. Washing was started with a 20 mM phosphatebuffer (pH 7.0) containing 0.3 M NaCl, and a flow-through fractioncorresponding to the start to the end of the peak of absorbance at 280nm was obtained as a roughly purified solution 2. The operation wasperformed at a temperature of 2 to 10° C., and a chromatography flowrate of 13 L/hour.

About 20 L of the roughly purified solution was applied to a monoclonalantibody column (diameter: 44 cm, height: 18 cm) equilibrated with a 20mM phosphate buffer (pH 7.3) containing 0.3 M sodium chloride. 6 CV of a20 mM phosphate buffer (pH 7.3) containing 1.0 M sodium chloride waspoured into the column, 3 CV of a 0.1 M acetate buffer (pH 5.0) wasfurther poured to wash the column, and elution was started with a 0.1 Mglycine-hydrochloric acid buffer (pH 3.0) containing 0.3 M sodiumchloride. The eluate corresponding to the start to the end of the peakof absorbance at 280 nm was obtained, and added with 1/10 volume of a 1M glycine-sodium hydroxide buffer (pH 9.0) and 1/25 volume of a 0.5 Mphosphate buffer (pH 7.3) to obtain a purified solution 1. The operationwas performed at a temperature of 2 to 10° C., and a chromatography flowrate of 50 L/hour.

About 15 L of the purified solution 1 was concentrated to about 1 L byusing an ultrafiltration membrane, Microza UF Module SIP-1013 (AsahiKasei Chemicals, Japan), and then applied to a Sephacryl S-300 HR (GEHealthcare Bioscience, U.S.A.) column (diameter: 25 cm, height: 80 cm)equilibrated with a 20 mM phosphate buffer (pH 7.3) containing 150 mMsodium chloride. The operation was performed at a temperature of 2 to10° C., and a chromatography flow rate of 1 L/hour. An elution peak withthe maximum absorbance at 280 nm was separated, and passed through a0.22-μm PVDF filtration membrane (Millipore, U.S.A.) to collect about 3L of the eluate. The result was used as a purified product of solublethrombomodulin (lot: B2).

The APC activity of thrombomodulin of B2 was 28000 U/mL.

Soluble thrombomodulin concentration in the solution of B2 was 3.8mg/mL.

Comparative Example 4 Preparation of Soluble Thrombomodulin 4

The cells of one vial cryopreserved in Comparative Example 1 werethawed, inoculated into a serum-free medium IS CHO-CD (IrvineScientific, U.S.A.) containing 8 mM L-glutamine (Invitrogen, U.S.A.), 50μM hypoxanthine (Invitrogen, U.S.A.), and 8 μM thymidine (Invitrogen,U.S.A.), and cultured at 37° C. in a CO₂ incubator. The cells obtainedby centrifuging the culture medium was suspended in the serum-freemedium IS CHO-CD (Irvine Scientific, U.S.A.) containing 8 mM L-glutamine(Invitrogen, U.S.A.), 50 μM hypoxanthine (Invitrogen, U.S.A.), 8 μMthymidine (Invitrogen, U.S.A.), and 10% dimethyl sulfoxide(Sigma-Aldrich, U.S.A.), and then the medium was dispensed into vials(2×10⁷ cells/vial), and cryopreserved in liquid nitrogen.

Growth medium was prepared by dissolving 20.78 g of IS CHO-CD-A3 (IrvineScientific, U.S.A.), 4.06 g of sodium chloride (Tomita Pharmaceutical,Japan), and 2.20 g of sodium hydrogencarbonate (Wako Pure ChemicalIndustries, Japan) in 1 L of water. Production medium was prepared bydissolving 20.78 g of IS CHO-CD-A3 (Irvine Scientific, U.S.A.), 2.63 gof sodium chloride (Tomita Pharmaceutical, Japan), and 4.40 g of sodiumhydrogencarbonate (Wako Pure Chemical Industries, Japan) in 1 L ofwater.

The cells of one vial were thawed, inoculated into 100 mL of the growthmedium, and cultured at 36° C. for 5 days as stationary culture by usinga T-flask in a CO₂ incubator. When the living cell density became7.0×10⁵ cells/mL or more, 40 mL of the culture medium was transferred to360 mL of the growth medium, and the cells were cultured with stirringat 36° C. for 7 days by using a spinner flask in a CO₂ incubator. Whenthe living cell density became 7.0×10⁵ cells/mL or more, 80 mL of theculture medium was transferred to 720 mL of the growth medium, and thecells were cultured with stirring at 36° C. for 6 days by using aspinner flask in a CO₂ incubator. When the living cell density became7.0×10⁵ cells/mL or more, the entire volume of the culture medium wastransferred to 9.2 L of the growth medium, and the cells were culturedwith stirring at 36° C., pH 7.1 and 50% of dissolved oxygen for 8 daysby using a perfusion culture tank. When the living cell density became7.0×10⁵ cells/mL or more, perfusion culture was started, in which theproduction medium was continuously added, and the culture supernatantwas continuously collected. The culture conditions consisted of 36° C.,pH 7.1, dissolved oxygen: 50%, medium exchange: 10 L/day, and surfacepressurization: 0 to 0.2 MPa. After the living cell density reached7.5×10⁶ cells/mL, the culture was further continued for 26 days, and theculture supernatant was collected as a production solution.

The collected production solution was clarified by using filtrationfilters, SUPRAcap (Pall, U.S.A.) and Supor EBV (Pall, U.S.A.), andstored at 2 to 10° C. as a filtered production solution.

About 250 L of the filtered production solution was applied to aQ-Sepharose Fast Flow (GE Healthcare, U.S.A.) column (diameter: 25 cm,height: 25 cm) equilibrated with a 20 mM Tris-hydrochloric acid buffer(pH 7.7) containing 150 mM sodium chloride. Then, the column was washedwith 6 CV of a 20 mM acetate buffer (pH 5.6) containing 180 mM sodiumchloride, and further washed with 4 CV of a 20 mM Tris-hydrochloric acidbuffer (pH 7.7) containing 180 mM sodium chloride. Elution was startedwith a 20 mM Tris-hydrochloric acid buffer (pH 7.7) containing 290 mMsodium chloride, and 0.5 column volume of the eluate from the start ofthe peak of absorbance at 280 nm was obtained as a roughly purifiedsolution. The operation was performed at a temperature of 2 to 10° C.,and a chromatography flow rate of 18 L/hour.

About 6 L of the roughly purified solution was applied to a monoclonalantibody column (diameter: 44 cm, height: 8 cm) equilibrated with a 20mM phosphate buffer (pH 7.3) containing 0.3 M sodium chloride. 6 CV of a20 mM phosphate buffer (pH 7.3) containing 1.0 M sodium chloride waspoured into the column, 3 CV of a 0.1 M acetate buffer (pH 5.0) wasfurther poured to wash the column, and elution was started with a 0.1 Mglycine-hydrochloric acid buffer (pH 3.0) containing 0.3 M sodiumchloride. The eluate corresponding to the start to the end of the peakof absorbance at 280 nm was obtained, and added with 1/10 volume of a0.5 M phosphate buffer (pH 7.3) to obtain a purified solution 1. Theoperation was performed at a temperature of 2 to 10° C., and achromatography flow rate of 46 L/hour.

About 14 L of the purified solution 1 was adjusted to pH 3.5 with a 1.0M glycine-hydrochloric acid buffer (pH 2.0), and applied to anSP-Sepharose FF (GE Healthcare Bioscience, U.S.A.) column (diameter: 14cm, height: 13 cm) equilibrated with a 0.1 M glycine-hydrochloric acidbuffer (pH 3.5) containing 0.3 M NaCl. Washing was started with a 0.1 Mglycine-hydrochloric acid buffer (pH 3.5) containing 0.3 M NaCl, and aflow-through fraction corresponding to the start to the end of the peakof absorbance at 280 nm was obtained, and immediately neutralized to pH7 with a 0.5 M phosphate buffer (pH 7.3) to obtain a purified solution2. The operation was performed at a temperature of 2 to 10° C., and achromatography flow rate of 15 L/hour.

About 20 L of the purified solution 2 was concentrated to about 1 L byusing an ultrafiltration membrane, Microza UF Module SIP-1013 (AsahiKasei Chemicals, Japan), and then applied to a Sephacryl S-300 HR (GEHealthcare Bioscience, U.S.A.) column (diameter: 25 cm, height: 79 cm)equilibrated with a 20 mM phosphate buffer (pH 7.3) containing 50 mMsodium chloride. An elution peak with the maximum absorbance at 280 nmwas separated, and concentrated to about 0.7 L by using anultrafiltration membrane, Microza UF Module SIP-1013 (Asahi KaseiChemicals, Japan), to obtain a purified solution 3. The operation wasperformed at a temperature of 2 to 10° C., and a chromatography flowrate of 1 L/hour.

The entire volume of the purified solution 3 was passed through avirus-removing membrane, PLANOVA 15N (membrane area: 0.12 m², AsahiKasei Medical, Japan), equilibrated with a 20 mM phosphate buffer (pH7.3) containing 50 mM sodium chloride at room temperature and a pressurelower than 0.1 MPa, and then further passed through a 0.22-μm PVDFfiltration membrane (Millipore, U.S.A.), and the entire volume of thesolution was collected. The result was used as a purified product ofsoluble thrombomodulin (lot: B3).

Example 1 Preparation of Highly-Purified Soluble Thrombomodulin 1

Cell culture was performed by using, as the base medium, the mediumdescribed in Japanese Patent Unexamined Publication No. 11-341990,Ingredient Table 2, provided that NaHCO₃ concentration was changed to5,700 mg/L, and NaCl concentration was changed to 2,410 mg/L. Growthmedium was obtained by adding 60 mg/L of kanamycin sulfate (Invitrogen,U.S.A.), 1 mg/L of tylosin tartrate (Sigma-Aldrich, U.S.A.), and 8%bovine serum (HyClone, U.S.A.) to the base medium, and used. Further,production medium was the same as the growth medium, provided that theserum concentration was changed to 3%.

The cells of one vial prepared in Comparative Example 1 were thawed,inoculated into 100 mL of the growth medium, and cultured with stirringat 37° C. for 5 days by using a spinner flask in a CO₂ incubator. Whenthe living cell density became 7.0×10⁵ cells/mL or more, the entirevolume of the culture medium was transferred to 0.9 L of the growthmedium, and the cells were cultured with stirring at 37° C. for 5 daysby using a spinner flask in a CO₂ incubator. When the living celldensity became 7.0×10⁵ cells/mL or more, the entire volume of theculture medium was transferred to 9 L of the growth medium, and thecells were cultured with stirring at 37° C., pH 7.2 and 50% of dissolvedoxygen for 5 days by using a culture tank. When the living cell densitybecame 7.0×10⁵ cells/mL or more, the entire volume of the culture mediumwas transferred to 120 L of the growth medium, and the cells werecultured with stirring at 37° C., pH 7.2 and 50% of dissolved oxygen for7 days by using a perfusion culture tank. When the living cell densitybecame 7.0×10⁵ cells/mL or more, perfusion culture was started, in whichthe production medium was continuously added, and the culturesupernatant was continuously collected. The culture conditions consistedof 37° C., pH 7.2, dissolved oxygen: 50%, medium exchange: 130 to 200L/day, and surface pressurization: 0 to 0.2 MPa. After the living celldensity reached 7.5×10⁶ cells/mL, the culture was further continued for40 days, and the culture supernatant was collected as a productionsolution. The collected production solution was clarified by usingfiltration filters, SUPRAdisc II (Pall, U.S.A.) and Supor EBV (Pall,U.S.A.), and stored at 2 to 10° C. as a filtered production solution.

About 700 L of the filtered production solution was applied to aQ-Sepharose Fast Flow (GE Healthcare, U.S.A.) column (diameter: 63 cm,height: 25 cm) equilibrated with a 20 mM Tris-hydrochloric acid buffer(pH 7.7) containing 150 mM sodium chloride. Then, the column was washedwith 6 CV of a 20 mM acetate buffer (pH 5.5) containing 180 mM sodiumchloride, and further washed with a 20 mM Tris-hydrochloric acid buffer(pH 7.7) containing 180 mM sodium chloride until absorbance at 280 nmreturned to the baseline. Elution was started with a 20 mMTris-hydrochloric acid buffer (pH 7.7) containing 300 mM sodiumchloride, and 0.5 column volume of the eluate from the start of the peakof absorbance at 280 nm was obtained as a roughly purified solution. Thesame operation was repeated 3 times to obtain 3 lots of the roughlypurified solution. The operation was performed at a temperature of 2 to10° C., and a chromatography flow rate of 109 L/hour.

About 20 L of the roughly purified solution was applied to a monoclonalantibody column (diameter: 44 cm, height: 13 cm) equilibrated with a 20mM phosphate buffer (pH 7.3) containing 0.3 M sodium chloride. 6 CV of a20 mM phosphate buffer (pH 7.3) containing 1.0 M sodium chloride waspoured into the column, 3 CV of a 0.1 M acetate buffer (pH 5.0) wasfurther poured to wash the column, and elution was started with a 0.1 Mglycine-hydrochloric acid buffer (pH 3.0) containing 0.3 M sodiumchloride. The eluate corresponding to the start to the end of the peakof absorbance at 280 nm was obtained, and added with 1/10 volume of a0.5 M phosphate buffer (pH 7.3) to obtain a purified solution 1. Thesame operation was repeated 6 times to obtain 6 lots of the purifiedsolution 1. The operation was performed at a temperature of 2 to 10° C.,and a chromatography flow rate of 46 L/hour.

About 130 L of the purified solution 1 of the 6 lots was passed througha nylon filtration membrane (pore diameter: 0.4 μm+0.2 μm, membranearea: 1.8 m², SARTOLON Maxi Caps 5101307H3, Sartorius, Germany) at aflow rate of 5 L/minute (about 0.07 m² of membrane area was used for 1mg of HCP), adjusted to pH 3.5 with a 1.0 M glycine-hydrochloric acidbuffer (pH 2.0), and applied to an SP-Sepharose FF (GE HealthcareBioscience, U.S.A.) column (diameter: 45 cm, height: 10 cm) equilibratedwith a 0.1 M glycine-hydrochloric acid buffer (pH 3.5) containing 0.3 MNaCl. Washing was started with a 0.1 M glycine-hydrochloric acid buffer(pH 3.5) containing 0.3 M NaCl, and a flow-through fractioncorresponding to the start to the end of the peak of absorbance at 280nm was obtained, and immediately neutralized to pH 7 with a 0.5 Mphosphate buffer (pH 7.3) to obtain a purified solution 2. The operationwas performed at a temperature of 2 to 10° C., and a chromatography flowrate of 160 L/hour.

About 160 L of the purified solution 2 was concentrated to about 10 L byusing an ultrafiltration membrane, Microza UF Module SIP-2013 (AsahiKasei Chemicals, Japan), and then applied to a Sephacryl S-300 HR (GEHealthcare Bioscience, U.S.A.) column (diameter: 63 cm, height: 94 cm)equilibrated with a 20 mM phosphate buffer (pH 7.3) containing 50 mMsodium chloride. An elution peak with the maximum absorbance at 280 nmwas separated, and concentrated to about 6 L by using an ultrafiltrationmembrane, Microza UF Module SIP-1013 (Asahi Kasei Chemicals, Japan), toobtain a purified solution 3. The operation was performed at atemperature of 2 to 10° C., and a chromatography flow rate of 6.2L/hour.

The purified solution 3 was passed through a virus-removing membrane,PLANOVA 15N (membrane area: 1 m², Asahi Kasei Medical, Japan),equilibrated with a 20 mM phosphate buffer (pH 7.3) containing 50 mMsodium chloride at room temperature and a pressure lower than 0.1 MPa,and then further passed through a 0.22-μm PVDF filtration membrane(Millipore, U.S.A.), and the entire volume of the solution wascollected. The result was used as a highly-purified solublethrombomodulin purified product (lot: A4).

The APC activity of thrombomodulin of A4 was 60000 U/mL.

Soluble thrombomodulin concentration in the solution of A4 was 9.3mg/mL.

Example 2 Preparation of Highly-Purified Soluble Thrombomodulin 2

About 2,000 L of the filtered production solution obtained in Example 1was applied to a Q-Sepharose Fast Flow (GE Healthcare, U.S.A.) column(diameter: 63 cm, height: 25 cm) equilibrated with a 20 mMTris-hydrochloric acid buffer (pH 7.7) containing 150 mM sodiumchloride. Then, the column was washed with 6 CV of a 20 mM acetatebuffer (pH 5.45) containing 170 mM sodium chloride, and further washedwith 4 CV of a 20 mM Tris-hydrochloric acid buffer (pH 7.7) containing170 mM sodium chloride. Elution was started with a 20 mMTris-hydrochloric acid buffer (pH 7.7) containing 300 mM sodiumchloride, and 0.5 column volume of the eluate from the start of the peakof absorbance at 280 nm was obtained as a roughly purified solution. Thesame operation was repeated twice to obtain 2 lots of the roughlypurified solution. The operation was performed at a temperature of 2 to10° C., and a chromatography flow rate of 109 L/hour.

About 10 L of the roughly purified solution was applied to a monoclonalantibody column (diameter: 44 cm, height: 13 cm) equilibrated with a 20mM phosphate buffer (pH 7.3) containing 0.3 M sodium chloride. 6 CV of a20 mM phosphate buffer (pH 7.3) containing 1.0 M sodium chloride waspoured into the column, 3 CV of a 0.1 M acetate buffer (pH 5.0) wasfurther poured to wash the column, and elution was started with a 0.1 Mglycine-hydrochloric acid buffer (pH 3.0) containing 0.3 M sodiumchloride. The eluate corresponding to the start to the end of the peakof absorbance at 280 nm was obtained, and added with 1/10 volume of a0.5 M phosphate buffer (pH 7.3) to obtain a purified solution 1. Thesame operation was repeated 8 times to obtain 8 lots of the purifiedsolution 1. The operation was performed at a temperature of 2 to 10° C.,and a chromatography flow rate of 46 L/hour.

About 180 L of the purified solution 1 for 6 lots was passed through anylon filtration membrane (pore diameter: 0.4 μm+0.2 μm, membrane area:1.8 m², SARTOLON Maxi Caps 5101307H3, Sartorius, Germany) at a flow rateof 5 L/minute (about 0.05 m² of membrane area was used for 1 mg of HCP),adjusted to pH 3.5 with a 1.0 M glycine-hydrochloric acid buffer (pH2.0), and applied to an SP-Sepharose FF (GE Healthcare Bioscience,U.S.A.) column (diameter: 45 cm, height: 10 cm) equilibrated with a 0.1M glycine-hydrochloric acid buffer (pH 3.5) containing 0.3 M NaCl.Washing was started with a 0.1 M glycine-hydrochloric acid buffer (pH3.5) containing 0.3 M NaCl, and a flow-through fraction corresponding tothe start to the end of the peak of absorbance at 280 nm was obtained,and immediately neutralized to pH 7 with a 0.5 M phosphate buffer (pH7.3) to obtain a purified solution 2. The operation was performed at atemperature of 2 to 10° C., and a chromatography flow rate of 160L/hour.

About 220 L of the purified solution 2 was concentrated to about 5 L byusing an ultrafiltration membrane, Microza UF Module SIP-2013 (AsahiKasei Chemicals, Japan), and then applied to a Sephacryl S-300 HR (GEHealthcare Bioscience, U.S.A.) column (diameter: 63 cm, height: 94 cm)equilibrated with a 20 mM phosphate buffer (pH 7.3) containing 50 mMsodium chloride. An elution peak with the maximum absorbance at 280 nmwas separated, and concentrated to about 10 L by using anultrafiltration membrane, Microza UF Module SIP-1013 (Asahi KaseiChemicals, Japan), to obtain a purified solution 3. The operation wasperformed at a temperature of 2 to 10° C., and a chromatography flowrate of 6.2 L/hour.

The purified solution 3 was passed through a virus-removing membrane,PLANOVA 15N (membrane area: 1 m², Asahi Kasei Medical, Japan),equilibrated with a 20 mM phosphate buffer (pH 7.3) containing 50 mMsodium chloride at room temperature and a pressure lower than 0.1 MPa,and then further passed through a 0.22-μm PVDF filtration membrane(Millipore, U.S.A.), and the entire volume of the solution wascollected. The result was used as a highly-purified solublethrombomodulin purified product (lot: A5).

The APC activity of thrombomodulin of A5 was 69000 U/mL.

Soluble thrombomodulin concentration in the solution of A5 was 10.9mg/mL.

Example 3 Preparation of Highly-Purified Soluble Thrombomodulin 3

Cell culture was performed by using, as the base medium, the mediumdescribed in Japanese Patent Unexamined Publication No. 11-341990,Ingredient Table 2, provided that NaHCO₃ concentration was changed to5,700 mg/L, and NaCl concentration was changed to 2,410 mg/L. Growthmedium was obtained by adding 60 mg/L of kanamycin sulfate (Invitrogen,U.S.A.), 1 mg/L of tylosin tartrate (Sigma-Aldrich, U.S.A.), and 8%bovine serum (HyClone, U.S.A.) to the base medium, and used. Further,production medium was the same as the growth medium, provided that theserum concentration was changed to 3%.

The cells of one vial prepared in Comparative Example 1 were thawed,inoculated into 100 mL of the growth medium, and cultured with stirringat 37° C. for 5 days by using a spinner flask in a CO₂ incubator. Whenthe living cell density became 7.0×10⁵ cells/mL or more, the entirevolume of the culture medium was transferred to 0.9 L of the growthmedium, and the cells were cultured with stirring at 37° C. for 5 daysby using a spinner flask in a CO₂ incubator. When the living celldensity became 7.0×10⁵ cells/mL or more, the entire volume of theculture medium was transferred to 9 L of the growth medium, and thecells were cultured with stirring at 37° C., pH 7.2 and 50% of dissolvedoxygen for 5 days by using a culture tank. When the living cell densitybecame 7.0×10⁵ cells/mL or more, the entire volume of the culture mediumwas transferred to 120 L of the growth medium, and the cells werecultured with stirring at 37° C., pH 7.2 and 50% of dissolved oxygen for7 days by using a perfusion culture tank. When the living cell densitybecame 7.0×10⁵ cells/mL or more, perfusion culture was started, in whichthe production medium was continuously added, and the culturesupernatant was continuously collected. The culture conditions consistedof 37° C., pH 7.2, dissolved oxygen: 50%, medium exchange: 130 to 200L/day, and surface pressurization: 0 to 0.2 MPa. After the living celldensity reached 7.5×10⁶ cells/mL, the culture was further continued for36 days, and the culture supernatant was collected as a productionsolution. The collected production solution was clarified by usingfiltration filters, SUPRAdisc II (Pall, U.S.A.) and Supor EBV (Pall,U.S.A.), and stored at 2 to 10° C. as a filtered production solution.

About 700 L of the filtered production solution was applied to aQ-Sepharose Fast Flow (GE Healthcare, U.S.A.) column (diameter: 63 cm,height: 25 cm) equilibrated with a 20 mM Tris-hydrochloric acid buffer(pH 7.7) containing 150 mM sodium chloride. Then, the column was washedwith 6 CV of a 20 mM acetate buffer (pH 5.5) containing 180 mM sodiumchloride, and further washed with a 20 mM Tris-hydrochloric acid buffer(pH 7.7) containing 180 mM sodium chloride until absorbance at 280 nmreturned to the baseline. Elution was started with a 20 mMTris-hydrochloric acid buffer (pH 7.7) containing 300 mM sodiumchloride, and 0.5 column volume of the eluate from the start of the peakof absorbance at 280 nm was obtained as a roughly purified solution. Thesame operation was repeated 6 times to obtain 6 lots of the roughlypurified solution. The operation was performed at a temperature of 2 to10° C., and a chromatography flow rate of 109 L/hour.

About 20 L of the roughly purified solution was applied to a monoclonalantibody column (diameter: 44 cm, height: 13 cm) equilibrated with a 20mM phosphate buffer (pH 7.3) containing 0.3 M sodium chloride. 6 CV of a20 mM phosphate buffer (pH 7.3) containing 1.0 M sodium chloride waspoured into the column, 3 CV of a 0.1 M acetate buffer (pH 5.0) wasfurther poured to wash the column, and elution was started with a 0.1 Mglycine-hydrochloric acid buffer (pH 3.0) containing 0.3 M sodiumchloride. The eluate corresponding to the start to the end of the peakof absorbance at 280 nm was obtained, and added with 1/10 volume of a0.5 M phosphate buffer (pH 7.3) to obtain a purified solution 1. Thesame operation was repeated 12 times to obtain 12 lots of the purifiedsolution 1. The operation was performed at a temperature of 2 to 10° C.,and a chromatography flow rate of 46 L/hour.

About 270 L of the purified solution 1 of the 12 lots was passed througha nylon filtration membrane (pore diameter: 0.4 μm+0.2 μm, membranearea: 1.8 m², SARTOLON Maxi Caps 5101307H3, Sartorius, Germany) at aflow rate of 5 L/minute (about 0.05 m² of membrane area was used for 1mg of HCP), adjusted to pH 3.5 with a 1.0 M glycine-hydrochloric acidbuffer (pH 2.0), and applied to an SP-Sepharose FF (GE HealthcareBioscience, U.S.A.) column (diameter: 45 cm, height: 10 cm) equilibratedwith a 0.1 M glycine-hydrochloric acid buffer (pH 3.5) containing 0.3 MNaCl. Washing was started with a 0.1 M glycine-hydrochloric acid buffer(pH 3.5) containing 0.3 M NaCl, and a flow-through fractioncorresponding to the start to the end of the peak of absorbance at 280nm was obtained, and immediately neutralized to pH 7 with a 0.5 Mphosphate buffer (pH 7.3) to obtain a purified solution 2. The operationwas performed at a temperature of 2 to 10° C., and a chromatography flowrate of 160 L/hour.

About 300 L of the purified solution 2 was concentrated to about 11 L byusing an ultrafiltration membrane, Microza UF Module SIP-2013 (AsahiKasei Chemicals, Japan), and then applied to a Sephacryl S-300 HR (GEHealthcare Bioscience, U.S.A.) column (diameter: 63 cm, height: 94 cm)equilibrated with a 20 mM phosphate buffer (pH 7.3) containing 50 mMsodium chloride. An elution peak with the maximum absorbance at 280 nmwas separated, and concentrated to about 13 L by using anultrafiltration membrane, Microza UF Module SIP-1013 (Asahi KaseiChemicals, Japan), to obtain a purified solution 3. The operation wasperformed at a temperature of 2 to 10° C., and a chromatography flowrate of 6.2 L/hour.

The purified solution 3 was passed through a virus-removing membrane,PLANOVA 15N (membrane area: 1 m², Asahi Kasei Medical, Japan),equilibrated with a 20 mM phosphate buffer (pH 7.3) containing 50 mMsodium chloride at room temperature and a pressure lower than 0.1 MPa,and then further passed through a 0.22-μm PVDF filtration membrane(Millipore, U.S.A.), and the entire volume of the solution wascollected. The result was used as a highly-purified solublethrombomodulin purified product (lot: A6).

The APC activity of thrombomodulin of A6 was 81000 U/mL.

Soluble thrombomodulin concentration in the solution of A6 was 11.9mg/mL.

Example 4 Preparation of Highly-Purified Soluble Thrombomodulin 4

Growth medium was prepared by dissolving 20.78 g of IS CHO-CD-A3 (IrvineScientific, U.S.A.), 4.06 g of sodium chloride (Tomita Pharmaceutical,Japan), and 2.20 g of sodium hydrogencarbonate (Wako Pure ChemicalIndustries, Japan) in 1 L of water. Production medium was prepared bydissolving 20.78 g of IS CHO-CD-A3 (Irvine Scientific, U.S.A.), 2.63 gof sodium chloride (Tomita Pharmaceutical, Japan), and 4.40 g of sodiumhydrogencarbonate (Wako Pure Chemical Industries, Japan) in 1 L ofwater.

The cells of one vial prepared in Comparative Example 4 were thawed,inoculated into 100 mL of the growth medium, and cultured at 36° C. for5 days as stationary culture by using a T-flask in a CO₂ incubator. Whenthe living cell density became 7.0×10⁵ cells/mL or more, the entirevolume of the culture medium was transferred to 0.9 L of the growthmedium, and the cells were cultured with stirring at 36° C. for 5 daysby using a spinner flask in a CO₂ incubator. When the living celldensity became 7.0×10⁵ cells/mL or more, the entire volume of theculture medium was transferred to 9 L of the growth medium, and thecells were cultured with stirring at 36° C., pH 7.1 and 50% of dissolvedoxygen for 5 days by using a culture tank. When the living cell densitybecame 7.0×10⁵ cells/mL or more, the entire volume of the culture mediumwas transferred to 120 L of the growth medium, and the cells werecultured with stirring at 36° C., pH 7.1 and 50% of dissolved oxygen for7 days by using a perfusion culture tank. When the living cell densitybecame 7.0×10⁵ cells/mL or more, perfusion culture was started, in whichthe production medium was continuously added, and the culturesupernatant was continuously collected. The culture conditions consistedof 36° C., pH 7.1, dissolved oxygen: 50%, medium exchange: 130 L/day,and surface pressurization: 0 to 0.2 MPa. After the living cell densityreached 7.5×10⁶ cells/mL, the culture was further continued for 20 days,and the culture supernatant was collected as a production solution.

The collected production solution was clarified by using filtrationfilters, SUPRAdisc II (Pall, U.S.A.) and Supor EBV (Pall, U.S.A.), andstored at 2 to 10° C. as a filtered production solution.

About 1,400 L of the filtered production solution was applied to aQ-Sepharose Fast Flow (GE Healthcare, U.S.A.) column (diameter: 63 cm,height: 25 cm) equilibrated with a 20 mM Tris-hydrochloric acid buffer(pH 7.7) containing 150 mM sodium chloride. Then, the column was washedwith 6 CV of a 20 mM acetate buffer (pH 5.6) containing 180 mM sodiumchloride, and further washed with 4 CV of a 20 mM Tris-hydrochloric acidbuffer (pH 7.7) containing 180 mM sodium chloride. Elution was startedwith a 20 mM Tris-hydrochloric acid buffer (pH 7.7) containing 290 mMsodium chloride, and 0.5 column volume of the eluate from the start ofthe peak of absorbance at 280 nm was obtained as a roughly purifiedsolution. The same operation was performed also for about 900 L of thefiltered production solution, and thus 2 lots of the roughly purifiedsolution were obtained. The operation was performed at a temperature of2 to 10° C., and a chromatography flow rate of 109 L/hour.

About 13 L of the roughly purified solution was applied to a monoclonalantibody column (diameter: 44 cm, height: 13 cm) equilibrated with a 20mM phosphate buffer (pH 7.3) containing 0.3 M sodium chloride. 6 CV of a20 mM phosphate buffer (pH 7.3) containing 1.0 M sodium chloride waspoured into the column, 3 CV of a 0.1 M acetate buffer (pH 5.0) wasfurther poured to wash the column, and elution was started with a 0.1 Mglycine-hydrochloric acid buffer (pH 3.0) containing 0.3 M sodiumchloride. The eluate corresponding to the start to the end of the peakof absorbance at 280 nm was obtained, and added with 1/10 volume of a0.5 M phosphate buffer (pH 7.3) to obtain a purified solution 1. Thesame operation was repeated 5 times to obtain 5 lots of the purifiedsolution 1. The operation was performed at a temperature of 2 to 10° C.,and a chromatography flow rate of 46 L/hour.

About 110 L of the purified solution 1 of the 5 lots was passed througha nylon filtration membrane (pore diameter: 0.4 μm+0.2 μm, membranearea: 1.8 m², SARTOLON Maxi Caps 5101307H3, Sartorius, Germany) at aflow rate of 5 L/minute (about 0.03 m² of membrane area was used for 1mg of HCP), adjusted to pH 3.5 with a 1.0 M glycine-hydrochloric acidbuffer (pH 2.0), and applied to an SP-Sepharose FF (GE HealthcareBioscience, U.S.A.) column (diameter: 45 cm, height: 10 cm) equilibratedwith a 0.1 M glycine-hydrochloric acid buffer (pH 3.5) containing 0.3 MNaCl. Washing was started with a 0.1 M glycine-hydrochloric acid buffer(pH 3.5) containing 0.3 M NaCl, and a flow-through fractioncorresponding to the start to the end of the peak of absorbance at 280nm was obtained, and immediately neutralized to pH 7 with a 0.5 Mphosphate buffer (pH 7.3) to obtain a purified solution 2. The operationwas performed at a temperature of 2 to 10° C., and a chromatography flowrate of 160 L/hour.

About 120 L of the purified solution 2 was concentrated to about 5 L byusing an ultrafiltration membrane, Microza UF Module SIP-2013 (AsahiKasei Chemicals, Japan), and then applied to a Sephacryl S-300 HR (GEHealthcare Bioscience, U.S.A.) column (diameter: 63 cm, height: 94 cm)equilibrated with a 20 mM phosphate buffer (pH 7.3) containing 50 mMsodium chloride. An elution peak with the maximum absorbance at 280 nmwas separated, and concentrated to about 5 L by using an ultrafiltrationmembrane, Microza UF Module SIP-1013 (Asahi Kasei Chemicals, Japan), toobtain a purified solution 3. The operation was performed at atemperature of 2 to 10° C., and a chromatography flow rate of 6.2L/hour.

The entire volume of the purified solution 3 was passed through avirus-removing membrane, PLANOVA 15N (membrane area: 1 m², Asahi KaseiMedical, Japan), equilibrated with a 20 mM phosphate buffer (pH 7.3)containing 50 mM sodium chloride at room temperature and a pressurelower than 0.1 MPa, and then further passed through a 0.22-μm PVDFfiltration membrane (Millipore, U.S.A.), and the entire volume of thesolution was collected. The result was used as a highly-purified solublethrombomodulin purified product (lot: A7).

The APC activity of thrombomodulin of A7 was 69000 U/mL.

Soluble thrombomodulin concentration in the solution of A7 was 10.4mg/mL.

Test Example 1 Evaluation of Removal of HCP Using Various FiltrationMembranes

The purified solution 1 obtained in Comparative Example 1 (HCPconcentration: 462 ng/ml) was passed through filtration membranes ofdifferent materials, and HCP concentrations of the filtrates werecompared. Specifically, 5 ml of the purified solution 1 was passed at aflow rate of 1 ml/minute through each of filtration membranes made of(1) PVDF (polyvinylidene fluoride) (Millex GV, Millipore, U.S.A.), (2)CA (cellulose acetate) (Minisart, Sartorius, Germany), (3) PES(polyethersulfone) (Minisart High-Flow, Sartorius, Germany), (4) nylon(NALGENE Syringe Filter, Thermo Fisher Scientific, U.S.A.), and (5)CA+GF (cellulose acetate+glass fiber) (Minisart Plus, Sartorius), andthe entire volume was collected as a filtrate.

Protein concentration and HCP concentration of the solution weremeasured before and after the filtration. The protein concentration wasobtained on the basis of absorbance at 280 nm, and the HCP concentrationwas measured according to the method described in Reference Example 2.As a result, substantial difference was not observed between the proteinconcentrations measured before and after the filtration for all thefiltration membranes, but high HCP-removing effect was observed for thefiltration membrane made of nylon and the filtration membrane made ofPES, and it was found that they reduced the HCP concentration to 28% and36%, respectively (Table 1). Further, degree of the reduction of the HCPconcentration significantly differed depending on the material of themembrane in spite of the same pore diameter. Accordingly, it was notconsidered that HCP insolubilized by aggregation was removed, but it wasconsidered that HCP was removed by adsorption by the membranes. Thediameters of the evaluated filtration membranes were 25 mm or 26 mm, andthe membrane areas for 1 mg of HCP calculated on the basis of effectivemembrane areas of the filtration membranes described in the data sheetsof the manufacturers were 0.17 m² or 0.23 m².

TABLE 1 Re- Re- Mem- Effective Membrane covery covery Pore branemembrane area for of of Filtration diameter diameter area 1 mg of HCPproteins membrane (μm) (mm) (cm²) HCP (m²) (%) (%) (1) PVDF 0.22 25 3.90.17 93 101 (2) CA 0.2  26 5.3 0.23 69 100 (3) PES 0.2  26 5.3 0.23 36100 (4) Nylon 0.2  25 Unknown Unknown 28  99 (5) CA + 0.2  26 5.3 0.2344  99 GF

Test Example 2 Comparison of HCP-Removing Abilities of Nylon and PESFiltration Membranes

It was found in Test Example 1 that the nylon filtration membrane andthe PES filtration membrane had high HCP-removing abilities.Accordingly, change of HCP-removing abilities of these filtrationmembranes depending on the volume of solution passed through them wasevaluated. Products of two manufacturers were prepared for each materialof the filtration membrane, and the purified solution 1 of a lotdifferent from the lots used in Test Example 1 was passed through themembranes (HCP concentration: 303 ng/ml). The filtration membranes usedwere a PVDF filtration membrane (pore diameter: 0.22 μm, membranediameter: 25 mm, effective membrane area: 3.9 cm², Millex GV, Millipore,U.S.A.) as a control, (1) Acrodisc AP-4436T, Pall, U.S.A., porediameter: 0.2 μm, membrane diameter: 25 mm, effective membrane area: 3.9cm², and (2) Minisart NY25, Sartorius, Germany, pore diameter: 0.2 μm,membrane diameter 25 mm, effective membrane area: 4.8 cm² as nylonfiltration membranes, as well as (3) Acrodisc PN4612, Pall, U.S.A., porediameter: 0.2 inn, membrane diameter 25 mm, effective membrane area: 2.8cm², and (4) Minisart High-Flow, Sartorius, Germany, pore diameter: 0.2μm, membrane diameter 26 mm, effective membrane area: 5.3 cm² as PESfiltration membranes. The purified solution 1 was passed through eachfiltration membrane in a volume of 100 ml at a flow rate of 10ml/minute, and the filtrate was sampled for every 20 ml of the solutionpassed through the filtration membrane, of which HCP concentration wasmeasured according to the method described in Reference Example 2, andof which protein concentration was obtained on the basis of absorbanceat 280 nm.

As a result, change of the protein concentration was not observed forall the samples, but HCP-removing effect was observed for both the nylonand PES filtration membranes (Table 2). The nylon filtration membranegave especially high HCP-removing effect, and reduced the HCPconcentration to 25% at most. It was found that when volume of thesolution passed through the filtration membrane was smaller, i.e.,filtration membrane area for 1 mg of HCP was larger, higher HCP-removingeffect was obtained.

TABLE 2 Membrane area for 1 mg of Recovery of HCP (%) Filtrationmembrane HCP (m²) 20 ml 40 ml 60 ml 80 ml 100 ml PVDF 0.013~0.064 94 98109 89  92 Nylon (1) Pall 0.013~0.064 50 62  60 69  75 (2) Sartorius0.016~0.079 25 34  30 49  47 PES (3) Pall 0.009~0.046 77 84  98 88 107(4) Sartorius 0.017~0.087 68 66  75 62  63

Test Example 3 Evaluation of HCP-Removing Ability of Nylon FiltrationMembrane for Different Solution Compositions

Effects of differences in buffer composition and soluble thrombomodulinconcentration on the HCP-removing ability of a nylon filtration membranewere evaluated. Each of the purified solution 1, the purified solution2, the purified solution 2 after the concentration, the purifiedsolution 3, and the purified product obtained in Comparative Example 4in a volume of 5 mL was passed through a nylon filtration membranehaving a membrane diameter of 25 mm, an effective membrane area of 4.8cm², and a pore diameter of 0.2 μm (Minisart NY25, Sartorius, Germany)at a flow rate of 1 mL/minute, and the entire volume was collected as afiltrate. The filtration membrane areas for 1 mg of HCP contained in thesamples were as follows: 0.77 m² for the purified solution 1, 1.7 m² forthe purified solution 2, 0.43 m² for the purified solution 2 after theconcentration, 0.72 m² for the purified solution 3, and 0.81 m² for thepurified product. HCP concentration of each obtained filtrate wasmeasured according to the method described in Reference Example 2, andprotein concentration of the same was obtained on the basis ofabsorbance at 280 nm. As a result, the nylon membrane gave highHCP-removing effect for all the samples, and provided high proteinrecovery higher than 90% (Table 3).

TABLE 3 HCP Protein Before After Re- Before After Re- filtrationfiltration covery filtration filtration covery Sample (ng/mL) (ng/mL)(%) (mg/mL) (mg/mL) (%) Purified 125 65 52 0.47 0.44 94 solution 1Purified 56 35 63 0.40 0.37 93 solution 2 Purified 222 115 52 8.28 8.28100 solution 2 after concentration Purified 134 <25 <19 11.1 11.4 103solution 3 Purified 119 <25 <21 10.1 10.3 102 product

Test Example 4 Evaluation of Removal of HCP from Purified Products ofSoluble Thrombomodulin Obtained by Various Preparation Methods

It was examined whether or not HCP was successfully removed from solublethrombomodulin products obtained by different methods by further passingthem through a nylon filtration membrane. The soluble thrombomodulinpurified products obtained in Comparative Examples 1 to 3 (A1, B1 andB2, respectively) in a volume of 5 mL each were passed through a nylonfiltration membrane having a membrane diameter 25 mm, an effectivemembrane area: 4.8 cm², and a pore diameter: 0.2 μm (Minisart NY25,Sartorius, Germany) at a flow rate of 1 mL/minute, and the entire volumeof each was collected as a filtrate. The filtration membrane areas for 1mg of HCP contained in the soluble thrombomodulin purified products were0.34 m² for A1, 0.46 m² for B1, and 1.7 m² for B2. HCP contents, mouseIgG contents, and bovine serum protein contents of the solutions weremeasured before and after the filtration by the methods described inReference Examples 2 to 4. The nylon membrane gave high removing effectfor only HCP for all the purified products (Table 4). On the basis ofthis result, it was considered that the nylon filtration membrane didnot have an action of non-specifically adsorbing proteins, but had anaction of specifically adsorbing HCP.

TABLE 4 Bovine serum HCP content Mouse IgG content protein content(ng/10,000 U) (ng/10,000 U) (ng/10,000 U) Purified Before After BeforeAfter Before After product filtration filtration filtration filtrationfiltration filtration Comparative 40.9 <8.2 <0.18 <0.18 0.67 1.03Example 1 (A1) Comparative 26.0 <6.7 <0.16 <0.16 0.24 <0.16 Example 2(B1) Comparative 20.0 7.0 0.65 0.59 13.5 13.2 Example 3 (B2)

Test Example 5 Comparison of Purities of Thrombomodulin PurifiedProducts Obtained with or without Use of Nylon Filtration Membrane

HCP contents, mouse IgG contents, and bovine serum protein contents ofthe thrombomodulin purified products obtained by the industrial levelproduction using no nylon filtration membrane (Comparative Example 1)and the industrial level production using a nylon filtration membrane(Examples 1 to 4) were measured by the methods described in ReferenceExamples 2 to 4.

The three lots of Comparative Example 1 (A1, A2 and A3) not passedthrough any nylon filtration membrane had high HCP contents higher than10 ng/10,000 U, whilst the HCP contents of the four lots of Examples 1to 4 (A4, A5, A6 and A7) passed through a nylon filtration membrane werelower than the quantification limit. Any significant difference was notobserved in contents of mouse IgG and bovine serum proteins as otherimpurities in the products obtained by using or not using the nylonfiltration membrane (Table 5). As described above, the nylon filtrationmembrane gave specific removing ability for HCP also in industrial levelproduction, and enabled production of highly-purified solublethrombomodulin having an HCP content less than 10 ng/10,000 U ofthrombomodulin.

Thrombomodulin purities based on the total proteins of thehighly-purified soluble thrombomodulin products obtained in Examples 1to 4 were measured by gel filtration liquid chromatography and ionexchange liquid chromatography. The measurement by gel filtration liquidchromatography was performed by using TOSOH TSKgel G3000SWXL (TOSOH,Japan), and a 50 mM phosphate buffer (pH 7.3) containing 0.1 M sodiumsulfate under the conditions of a temperature of 40° C. and a flow rateof 0.9 mL/minute. As a result, purities of the purified products (A4,A5, A6 and A7) were all higher than 99%. Further, the measurement by ionexchange liquid chromatography was performed by using TOSOH DEAE 5PW(TOSOH, Japan) with elution using a linear gradient of from a 20 mMpiperazine-hydrochloric acid buffer (pH 5.6) containing 50 mM sodiumchloride to a 20 mM piperazine-hydrochloric acid buffer (pH 5.6)containing 350 mM sodium chloride over 30 minutes under the conditionsof a temperature of 40° C. and a flow rate of 0.9 mL/minute. As aresult, the purities of the purified products (A4, A5, A6 and A7) wereall higher than 99%.

Further, when molecular weights of a soluble thrombomodulin purifiedproduct prepared as described in Comparative Example 1, of whichmolecular weight had been already confirmed to be 64,000 byMALDI-TOF-MS, and the highly-purified soluble thrombomodulin purifiedproducts obtained in Examples 1 to 4 were compared by SDS-PAGE, thebands were detected at the same position. On the basis of this result,the molecular weight of the highly-purified soluble thrombomodulin wasconsidered to be 64,000.

Furthermore, endotoxin contents determined by the gelling methoddescribed in Japanese Pharmacopoeia, General Test Procedures, EndotoxinTest Method <4.01> were 0.004 to 0.03 EU/10,000 U, and thus were at anextremely low level (Table 6).

TABLE 5 Application Mouse Bovine serum or non- HCP IgG proteinapplication content content content Lot of filtration (ng/10⁴ U) (ng/10⁴U) (ng/10⁴ U) Comparative A1 Not used 40.9 <0.18 0.67 Example 1Comparative A2 Not used 17.2 <0.19 0.25 Example 1 Comparative A3 Notused 22.3 <0.18 <0.23 Example 1 Example 1 A4 Used <8.3 <0.21 1.42Example 2 A5 Used <7.2 <0.18 0.42 Example 3 A6 Used <6.2 <0.16 0.71Example 4 A7 Used <7.2 <0.18 Not measured

TABLE 6 Application or non- Endotoxin content Lot application offiltration (EU/10⁴ U) Example 1 A4 Used 0.0050 Example 2 A5 Used 0.0043Example 3 A6 Used 0.0105 Example 4 A7 Used 0.0245

Test Example 6 Analysis of HCP Removed with Nylon Filtration Membrane

Highly-purified soluble thrombomodulin was prepared in the same manneras that described in Example 3, and the nylon filtration membrane usedfor the preparation (pore diameter: 0.4 μm+0.2 μm, membrane area: 1.8m², SARTOLON Maxi Caps 5101307H3, Sartorius, Germany) was taken out fromthe housing, and cut into pieces of about 3 g each. Five of the pieceswere sufficiently washed with a 20 mM phosphate buffer (pH 7.3)containing 50 mM sodium chloride, and then each piece was put into atest tube containing 40 mL of a 50 mM Tris-hydrochloric acid buffer (pH8.0) containing 0.5% CHAPS and 200 mM sodium chloride. The proteinsadsorbed on the membrane were extracted in the buffer by shakingovernight at room temperature. The entire volume of the extract wasconcentrated to 15 μL by using ultrafiltration membranes, Vivaspin 20(Sartorius, Germany) and Amicon Ultra-0.5 mL (Millipore, U.S.A.). ⅕Volume of this concentrate was subjected to SDS-PAGE (e-PAGEL5/20, Atto,Japan) and CBB staining (Quick-CBB, Wako Pure Chemical Industries,Japan). A band detected around a molecular weight of 10,000 was excised,and the gel portion was reduced with dithiothreitol, thencarbamidomethylated with iodoacetamide, and subjected to enzymaticdigestion with trypsin overnight. The enzymatic digestion product wassubjected to LC/MS/MS, and Mascot search was performed on the basis ofthe obtained mass-spectrum data by using the database of NCBI to analyzethe amino acid sequence of the enzymatic digestion product.

Measurement Conditions of LC/MS/MS

LC/MS/MS (measurement apparatus): DiNa-2A Multi-dimensional AutoinjectorSystem (KYA Technologies, Japan)MS measurement range: MS1 (m/z 400-1500), MS2 (m/z 50-1500)×3 (datadependent scanning mode)Ionization mode: nanoESI⁺

Column: PicoFrit Column BataBasic C18 (New Objective, U.S.A.) MobilePhase:

Mobile phase A: 0.1% formic acid/2% acetonitrileMobile phase B: 0.1% formic acid/80% acetonitrileGradient: 0 to 30 minutes: Mobile phase B, 5 to 40%

-   -   30 to 40 minutes: Mobile phase B, 40 to 100%    -   40 to 60 minutes: Mobile phase B, 100%        Flow rate: 300 nL/minute

The amino acid sequences of the fragments expected from themass-spectrum data were as shown in (1) to (7) mentioned below, andagreed with partial sequences of histone H2B (Biochimie, 61 (1), 61-69(1979)) shown below. This result revealed that one of constituents ofHCP removed with the nylon filter was histone H2B.

(SEQ ID NO: 14) (1) KESYSVYVYK (SEQ ID NO: 15) (2) VLKQVHPDTGISSK(SEQ ID NO: 16) (3) STITSREIQTAVR (SEQ ID NO: 17) (4) EIQTAVR(SEQ ID NO: 18) (5) EIQTAVRLLLPGELAK (SEQ ID NO: 19) (6) LLLPGELAK(SEQ ID NO: 20) (7) LLLPGELAKHAVSEGTK

TABLE 7 Amino acid sequence of histone H2B  (SEQ ID NO: 21)

(The boxed sequences are amino acid sequence regions of the amino acidsequence of histone H2B identical with the amino acid sequences of (1)to (7) mentioned above, which were deduced from the mass-spectrum data.)

What is claimed is:
 1. Highly-purified soluble thrombomodulin which hasa content of host cell-originated proteins being in a ratio of less than10 ng of the proteins per 10,000 U of the soluble thrombomodulin,wherein the soluble thrombomodulin is produced by a transformant cellobtained by transfecting a host cell with a DNA containing a nucleotidesequence encoding the soluble thrombomodulin.
 2. The highly-purifiedsoluble thrombomodulin according to claim 1, which is manufactured on anindustrial scale.
 3. The highly-purified soluble thrombomodulinaccording to claim 1 or 2, which is used as a material for a medicament.4. The highly-purified soluble thrombomodulin according to claim 1 or 2,wherein purity of the highly-purified soluble thrombomodulin is 99% orhigher based on the total proteins.
 5. The highly-purified solublethrombomodulin according to claim 1 or 2, wherein the solublethrombomodulin is produced by serum-free culture of the transformantcell.
 6. The highly-purified soluble thrombomodulin according to claim 1or 2, wherein the host cell is a Chinese hamster ovary cell.
 7. Thehighly-purified soluble thrombomodulin according to claim 1 or 2,wherein the soluble thrombomodulin has the following properties (1) to(5): (1) an action of selectively binding to thrombin, (2) an action ofpromoting activation of Protein C by thrombin, (3) an action ofextending thrombin clotting time, (4) an action of suppressing plateletaggregation caused by thrombin, and (5) anti-inflammatory action.
 8. Thehighly-purified soluble thrombomodulin according to claim 1 or 2,wherein molecular weight of the soluble thrombomodulin is in the rangeof 50,000 to 80,000.
 9. The highly-purified soluble thrombomodulinaccording to claim 1 or 2, wherein the highly-purified solublethrombomodulin is produced by a method comprising the following steps:(a) the step of obtaining a transformant cell by transfecting a hostcell with a DNA encoding soluble thrombomodulin; (b) the step ofobtaining a solution containing soluble thrombomodulin by culturing thetransformant cell, and (c) the step of bringing the solution containingsoluble thrombomodulin into contact with nylon and/or polyethersulfoneto obtain highly-purified soluble thrombomodulin having a content ofhost cell-originated proteins being in a ratio of less than 10 ng of theproteins per 10,000 U of soluble thrombomodulin.
 10. The highly-purifiedsoluble thrombomodulin according to claim 1 or 2, wherein the solublethrombomodulin is a peptide containing: (i) the amino acid sequence ofthe positions 367 to 480 in the amino acid sequence of SEQ ID NO: 9 or11, and the amino acid sequence of (ii-1) or (ii-2) mentioned below, andthe peptide is soluble thrombomodulin having the following properties(1) to (5): (ii-1) the amino acid sequence of the positions 19 to 244 inthe amino acid sequence of SEQ ID NO: 9 or 11, or (ii-2) the amino acidsequence of (ii-1) mentioned above, further including substitution,deletion or addition of one or more amino acid residues, (1) an actionof selectively binding to thrombin, (2) an action of promotingactivation of Protein C by thrombin, (3) an action of extending thrombinclotting time, (4) an action of suppressing platelet aggregation causedby thrombin, and (5) anti-inflammatory action.
 11. The highly-purifiedsoluble thrombomodulin according to claim 1 or 2, wherein the solublethrombomodulin is a peptide containing: (i-1) the amino acid sequence ofthe positions 19 to 516 in the amino acid sequence of SEQ ID NO: 9 or11, or (i-2) the amino acid sequence of (i-1) mentioned above, furtherincluding substitution, deletion or addition of one or more amino acidresidues, and the peptide is soluble thrombomodulin having theproperties (1) to (5) mentioned below: (1) an action of selectivelybinding to thrombin, (2) an action of promoting activation of Protein Cby thrombin, (3) an action of extending thrombin clotting time, (4) anaction of suppressing platelet aggregation caused by thrombin, and (5)anti-inflammatory action.
 12. The highly-purified soluble thrombomodulinaccording to claim 1 or 2, wherein the DNA containing a nucleotidesequence encoding soluble thrombomodulin is a DNA encoding the aminoacid sequence of SEQ ID NO: 9 or
 11. 13. A pharmaceutical compositioncomprising the highly-purified soluble thrombomodulin according to claim1 or 2 and a pharmaceutically acceptable carrier.